Immunomodulatory compounds and methods of use thereof

ABSTRACT

The present invention is directed to novel compounds that function as immunological adjuvants when co-administered with antigens such as vaccines for bacterial and viral diseases, to novel adjuvant formulations which include at least one of the adjuvant compounds of the invention, to novel immunostimulatory compositions which comprise an antigen and at least one of the adjuvant compounds of the invention, and to methods for the immunization of an animal by co-administration of a compound of the invention with an antigen against which the animal is to be immunized.

BACKGROUND OF THE INVENTION

Vaccines have proven to be successful, highly acceptable methods for theprevention of infectious diseases. They are cost effective, and do notinduce antibiotic resistance to the target pathogen or affect normalflora present in the host. In many cases, such as when inducinganti-viral immunity, vaccines can prevent a disease for which there areno viable curative or ameliorative treatments available.

Vaccines function by triggering the immune system to mount a response toan agent, or antigen, typically an infectious organism or a portionthereof that is introduced into the body in a non-infectious ornon-pathogenic form. Once the immune system has been “primed” orsensitized to the organism, later exposure of the immune system to thisorganism as an infectious pathogen results in a rapid and robust immuneresponse that destroys the pathogen before it can multiply and infectenough cells in the host organism to cause disease symptoms.

The agent, or antigen, used to prime the immune system can be the entireorganism in a less infectious state, known as an attenuated organism, orin some cases, components of the organism such as carbohydrates,proteins or peptides representing various structural components of theorganism.

In many cases, it is necessary to enhance the immune response to theantigens present in a vaccine in order to stimulate the immune system toa sufficient extent to make a vaccine effective, i.e., to conferimmunity. Many protein and most peptide and carbohydrate antigens,administered alone, do not elicit a sufficient antibody response toconfer immunity. Such antigens need to be presented to the immune systemin such a way that they will be recognized as foreign and will elicit animmune response. To this end, additives (adjuvants) have been devisedwhich immobilize antigens and stimulate the immune response.

The best known adjuvant, Freund's complete adjuvant, consists of amixture of mycobacteria in an oil/water emulsion. Freund's adjuvantworks in two ways: first, by enhancing cell and humoral-mediatedimmunity, and second, by blocking rapid dispersal of the antigenchallenge (the “depot effect”). However, due to frequent toxicphysiological and immunological reactions to this material, Freund'sadjuvant cannot be used in humans.

Another molecule that has been shown to have immunostimulatory oradjuvant activity is endotoxin, also known as lipopolysaccharide (LPS).LPS stimulates the immune system by triggering an “innate” immuneresponse—a response that has evolved to enable an organism to recognizeendotoxin (and the invading bacteria of which it is a component) withoutthe need for the organism to have been previously exposed. While LPS istoo toxic to be a viable adjuvant, molecules that are structurallyrelated to endotoxin, such as monophosphoryl lipid A (“MPL”) are beingtested as adjuvants in clinical trials. Currently, however, the onlyFDA-approved adjuvant for use in humans is aluminum salts (Alum) whichare used to “depot” antigens by precipitation of the antigens. Alum alsostimulates the immune response to antigens.

Thus, there is a recognized need in the art for compounds which can beco-administered with antigens in order to stimulate the immune system togenerate a more robust antibody response to the antigen than would beseen if the antigen were injected alone or with Alum.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to novel compounds thatfunction as immunological adjuvants when co-administered with antigenssuch as vaccines for bacterial and viral diseases.

In a second aspect, the present invention is directed to novel adjuvantformulations which comprise at least one of the adjuvant compounds ofthe invention.

In a third aspect, the invention is directed to novel immunostimulatorycompositions which comprise an antigen and at least one of the adjuvantcompounds of the invention.

In another aspect, the present invention is directed to methods for theimmunization of an animal by co-administration of a compound of theinvention with an antigen against which the animal is to be immunized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows the results of an in vitro assay forinduction of TNF-alpha cytokine release by compounds 100, 184 or 186 ofthe invention.

FIG. 2 is a graph that shows stimulation of alkaline phosphataseexpression from an inducible reporter construct with the TNF promoter(TNF-PLAP) in THP-1 cells by compounds 106 and 126 in the absence andpresence of 10% serum.

FIG. 3 is a graph showing stimulation of IL-10 release from normal mousesplenocytes by compounds 104, 106, 124, 126, 160, and 162 of theinvention.

FIG. 4 is a graph showing stimulation of interferon-gamma release fromnormal mouse splenocytes by compounds 104, 106, 124, 126, 160, and 162of the invention.

FIG. 5 is a graph illustrating the results of serum tritration analysisfor determining the amounts of antibody that are produced in response tokeyhole limpet hemocyanin in the absence and presence of compounds 100,124, and 126 of the invention.

FIG. 6 is a graph illustrating the results of serum titration analysisfor determining amounts of antibody produced in response to tetanustoxoid in the absence and presence of compounds 100, 116, 126, 160 and184 of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed in part to the novel compounds of theformulae I, II, III, and IV; immunological adjuvant formulationscomprising a compound of formula I, II, III, and IV; and at least oneadditional component; methods of using the compounds of the formula I,II, III, and IV; and methods of using the immunological formulationscomprising compounds of the formula I, II, III, and IV and at least oneadditional component.

Wherein for each of formula I, II, or III:

R¹ is selected from the group consisting of

(a) C(O);

(b) C(O)—C₁₋₁₄ alkyl-C(O), wherein said C₁₋₁₄ alkyl is optionallysubstituted with hydroxy, C₁₋₅ alkoxy, C₁₋₅ alkylenedioxy, C₁₋₅alkylamino, or C₁₋₅-alkyl-aryl, wherein said aryl moiety of saidC₁₋₁₅-alkyl-aryl is optionally substituted with C₁₋₅ alkoxy, C₁₋₅ alkylamino, C₁₋₅ alkoxy-amino, C₁₋₅ alkylamino-C₁₋₅ alkoxy, —O—C₁₋₅alkylamino-C₁₋₅ alkoxy, —O—C₁₋₅ alkylamino-C(O)—C₁₋₅ alkyl C(O)OH,—O—C₁₋₅ alkylamino-C(O)—C₁₋₅ alkyl-C(O)—C₁₋₅ alkyl;

(c) C₂ to C₁₅ straight or branched chain alkyl optionally substitutedwith hydroxy or alkoxy; and

(d) —C(O)—C₆₋₁₂ arylene-C(O)— wherein said arylene is optionallysubstituted with hydroxy, halogen, nitro or amino;

a and b are independently 0, 1, 2, 3 or 4;

d, d′, d″, e, e′ and e″ are independently an integer from 0 to 4;

X¹, X², Y¹, and Y² are independently selected from the group consistingof null, oxygen, NH and N(C(O)C₁₋₄ alkyl), and —N(C₁₋₄ alkyl)-;

W¹ and W² are independently selected from the group consisting ofcarbonyl, methylene, sulfone and sulfoxide;

R² and R⁵ are independently selected from the group consisting of:

(a) C₂ to C₂₀ straight chain or branched chain alkyl which is optionallysubstituted with oxo, hydroxy or alkoxy,

(b) C₂ to C₂₀ straight chain or branched chain alkenyl or dialkenylwhich is optionally substituted with oxo, hydroxy or alkoxy;

(c) C₂ to C₂₀ straight chain or branched chain alkoxy which isoptionally substituted with oxo, hydroxy or alkoxy;

(d) —NH—C₂ to C₂₀ straight chain or branched chain alkyl, wherein saidalkyl group is optionally substituted with oxo, hydroxy or alkoxy; and

(e)

wherein Z is selected from the group consisting of O and NH, and M and Nare independently selected from the group consisting of C₂ to C₂₀straight chain or branched chain alkyl, alkenyl, alkoxy, acyloxy,alkylamino, and acylamino; R¹and R⁶ are independently selected from thegroup consisting of C₂ to C₂₀ straight chain or branched chain alkyl oralkenyl optionally substituted with oxo or fluoro;

R⁴ and R⁷ are independently selected from the group consisting of C(O)C₂to C₂₀ straight chain or branched chain alkyl or alkenyl; C₂ to C₂₀straight chain or branched chain alkyl; C₂ to C₂₀ straight chain orbranched chain alkoxy; C₂ to C₂₀ straight chain or branched chainalkenyl; wherein said alkyl, alkenyl or alkoxy groups can beindependently and optionally substituted with hydroxy, fluoro or C₁ toC₅ alkoxy;

G¹, G², G³ and G⁴ are independently selected from the group consistingof oxygen, methylene, amino, thiol, —O—C(O)—, —NHC(O)—, —C(O)NH—, and—N(C(O)C₁₋₄ alkyl)-;

or G²R⁴ or G⁴R⁷ may together be a hydrogen atom or hydroxyl;

or a pharmaceutically acceptable salt thereof;

and wherein for Formula II:

a′ and b′ are independently 2, 3, 4, 5, 6, 7, or 8, preferably 2;

Z¹ is selected from the group consisting of —OP(O)(OH)₂, —P(O)(OH)₂,—OP(O)(OR⁸)(OH) where R⁸ is a C1-C4 alkyl chain, —OS(O)₂OH, —S(O)₂OH—,—CO₂H, —OB(OH)₂, —OH, —CH₃, —NH₂, NR⁹ ₃ where R⁹ is a C1-C4 alkyl chain;

Z² is —OP(O)(OH)₂, —P(O)(OH)_(2′), —OP(O)(OR¹⁰)(OH) where R¹⁰ is a C₁-₄alkyl chain, —OS(O)₂OH, —S(O)₂OH, CO₂H, —OB(OH)₂, —OH, CH₃, —NH₂, —NH¹¹,where R¹¹ is a C₁-₄ alkyl chain;

and wherein for Formula III:

R¹² is selected from H and a C₁-₄ alkyl chain;

and wherein for Formula 3:

R¹² is selected from H and a C1-C4 alkyl chain;

or a pharmaceutical salt thereof,

with the proviso that the compounds of formula I, II, or III are not

In preferred compounds of the invention, one or more of the following ispresent: each of a and b is 2; each of X¹ and Y¹ is NH; R¹ is C(O) orC(O)—C₁₋₁₄ alkyl-C(O); each of d′ and e′ is 1; each of d″ and e″ is 1; Xis O or NH, more preferably NH; and W is C(O); or each of d′ and e′ are2.

In further preferred embodiments, R¹ is a C(O)C₁₋₁₄ alkyl-C(O), whereinsaid C₁₋₁₄ alkyl is substituted, for example with a C₁₋₅ alkoxy group;

In another embodiment, the invention provides compounds of formula IV,which are described in U.S. Application No. 60/118,131 filed Feb. 1,1999, to which the present application claims priority. The compound offormula IV is:

wherein R¹ is selected from the group consisting of C₁ carbonyl, C₂ toC₁₅ discarbonyl optionally substituted with hydroxy or alkoxy, and C₂ toC₁₅ straight or branched chain alkyl optionally substituted with hydroxyor alkoxy;

wherein each of a and b is independently an integer from 2 to 4;

wherein each of d and e is independently an integer from 1 to 4;

wherein each of X and Y is independently selected from the groupconsisting of oxygen, and NH;

wherein R² and R⁵ are independently selected from the group consistingof:

(a) C₂ to C₂₀ straight chain or branched chain alkyl, wherein said C₂ toC₂₀ straight chain or branched chain alkyl is optionally substitutedwith hydroxy or alkoxy,

(b) C₂ to C₂₀ straight chain or branched chain alkenyl, wherein said C₂to C₂₀ straight chain or branched chain alkenyl is optionallysubstituted with hydroxy or alkoxy;

(c) CH₂-alkyl carbonyl;

(d) CH₂-alkenyl carbonyl; and

(e)

wherein Z is selected from the group consisting of O and NH, and M and Nare independently selected from the group consisting of C₂ to C₂₀straight chain or branched chain alkyl, alkenyl, alkoxy, acyloxy,alkylamino, and acylamino;

wherein R³ and R⁶ are independently selected from the group consistingof C₂ to C₂₀ straight chain or branched chain alkyl and C₂ to C₂₀straight chain or branched chain alkenyl;

wherein R⁴ and R⁷ are independently selected from the group consistingof hydrogen, C₂ to C₂₀ straight chain or branched chain alkyl, C₂ to C₂₀straight chain or branched chain alkenyl, C₂ to C₂₀ straight chain orbranched chain alkyl carbonyl, and C₂ to C₂₀ straight chain or branchedchain alkenyl carbonyl wherein said C₂ to C₂₀ straight chain or branchedchain alkyl and C₂ to C₂₀ straight chain or branched chain alkenyl canbe indepedently and optionally substituted with hydroxy or alkoxy;

or a pharmaceutically acceptable salt thereof;

with the proviso that the compound of formula IV is not

In yet another embodiment R¹ is C(O) and d and e are 1.

In a further embodiment, the invention is directed to compounds 100 (ER112022), 104 (ER 111231), 106 (ER 111232), 124 (ER 112065), 126 (ER112066), 160 (ER 113651), and 184 (ER 119327), their pharmaceuticallyacceptable salts, and compositions containing these compounds.

In a most preferred embodiment, the invention is directed to compoundsER 803022, ER 803058, ER 803732, ER 804053, ER 804057, ER 804058, ER804059, ER 804442, ER 804680 and ER 804764, and compositions containingthese compounds.

The invention is also directed to novel immunostimulatory compositionswhich include an antigen and an immunological adjuvant formulation ofthe invention as disclosed above.

Also provided are methods for the immunization of an animal byco-administration of a compound of the invention or an adjuvantformulation of the invention with an antigen against which the animal isto be immunized.

Definitions

Carbonyl, as used herein, is a (C═O) moiety.

Dicarbonyl, as used herein, is a moiety with the structure(C═O)-alkyl-(C═O) or (C═O)-aryl-(C═O), which is bonded to a moleculethrough the carbon atoms of both of the terminal carbonyl moieties.

Oxo, as used herein, is a ═O group.

Alkyl ester, as used herein, is a moiety with the structureO—(C═O)-alkyl, which is bonded to a molecule through the non-doublebonded oxygen of the ester group.

Alkenyl ester, as used herein, is a moiety with the structureO—(C═O)-carbon chain, where the carbon chain contains a carbon-to-carbondouble bond, which is bonded to a molecule through the non-double bondedoxygen of the ester group.

The term “alkylene” means a bivalent straight chain or branched alkylhydrocarbon group.

The term “alkenylene” means a bivalent straight chain or branchedhydrocarbon group having a single carbon to carbon double bond.

The term “dialkenylene” means a bivalent unsaturated straight chain orbranched chain hydrocarbon group having two carbon to carbon doublebonds.

The term “arylene” refers to a bivalent aromatic group.

The abbreviation “Boc” as used herein means t-butyloxycarbonyl.

As used herein with reference to compounds and compositions of theinvention, the term “type 1” refers to those compounds of the inventioncorresponding to formula I above where the values of a and b are thesame; the values of d and e are the same; the values of d′ and e′ arethe same; the values of d″ and e″ are the same; X¹ and Y¹ are the same;X² and Y² are the same; W¹ and W² are the same; R² and R⁵ are the same;G¹ and G³ are the same; R³ and R⁶ are the same; G² and G⁴ are the same;and R⁴ and R⁷ are the same. “Type 2”, as used herein, refers tocompounds or compositions corresponding to formula I where any one ormore of the following applies: the values of a and b are different, thevalues of d and e are the same, the values of d′ and e′ are different;the values of d″ and e″ are the same; X¹ and Y¹ are different; X² and Y²are different; W¹ and W² are different; R² and R⁵ are different; G¹ andG³ are different; R³ and R⁶ are different; G² and G⁴ are different; orR⁴ and R⁷ are different.

All patents, patent applications, and publications referred to hereinare incorporated by reference in their entirety. In case of a conflictin terminology, the present specification is controlling.

General Synthetic Methods

1. Synthesis of Diamide Compounds

In general, a 2-amino-1,3-dihydroxypropane or (±) serinol is transformedinto the 2-azido compound by reaction with trifluoromethanesulfonylazide followed by protection as the per-acetate for easy manipulation.The resulting compound is deacetylated, followed by reaction with anappropriately activated primary alcohol of a diol moiety. The primaryalcohol moiety of the product of this reaction is then protected, e.g.,by using TBDPSCL, followed by reaction with phosgene and then allylalcohol, to yield a fully protected diol. The protected diol is thentreated to cleave the protecting group from the primary alcohol. Theunprotected alcohol is reacted with a properly functionalizedphosphorylating reagent with formula (11) as indicated in the Examples,to form a phosphate ester compound. The azido moiety of the product isreduced, and then reacted with an activated acyl acid to form an amide.The protected terminal amine on the functionalized phosphate isdeprotected, and subsequently reacted with a phosgene or a dicarboxylicacid in the presence of a dehydrating agent, such as EDC. The phosphategroups of the resulting compound are then deprotected, yielding aracemic amide.

2. Synthesis of Chiral Diamide Compounds of Type 1

In general, a chiral amino acid ester with the desired structure isprotected with a benzimidate ester. The protected compound is reactedwith a reducing agent, e.g., DIBAL or the like, to reduce the acidmoiety of the amino acid to an alcohol. The resulting alcohol compoundis reacted with an appropriately activated primary alcohol of a diolmoiety, followed by cleavage of the benzimidate protecting group,yielding an amino-diol. The diol is then reacted with an appropriateacid chloride to yield a diol-amide.

The diol-amide is then reacted with a properly functionalizedphosphorylating reagent at the free primary hydroxyl group. Theresulting compound is esterified at the secondary alcohol group with anappropriate acyl moiety. The N-BOC group is then cleaved from the aminogroup introduced by phosphorylating reagent (11), yielding a phosphateester compound with a free primary amine. This product is then reactedwith phosgene or a dicarboxylic acid in the presence of a dehydratingagent, to yield a diamide product. The protected phosphate groups of thediamide product are then deprotected, typically with palladium(0) andphenylsilane.

3. Synthesis of Chiral Diamide Compounds of Type 2

Chiral diamide compounds of Type 2 are synthesized essentially asdescribed for chiral diamide compounds of Type 1, up to the point justafter cleavage of the protecting group from the primary amine group ofthe phosphate ester compound. At this point, a dicarboxylic acid whichhas one of the acid moieties protected is reacted with the primary aminegroup, to yield a monoamide. The protecting group on the othercarboxylic acid is then cleaved, providing a free carboxylic acid whichcan then be reacted with a primary amine from an alternative,appropriately substituted phosphate system, in the presence of adehydrating agent to yield a diamide of type 2, which can then betreated to deprotect the phosphate group or groups to yield a desiredcompound of the invention.

In the special case of chiral urea compounds of type 2 of the invention,the primary amino group of the N-BOC amino group of the phosphate esteris deprotected and then reacted with trichloromethyl chloroformate orthe like, in order to form an isocyanate compound. The isocyanate isthen reacted with a primary amine from an alternative, appropriatelysubstituted phosphate system to yield a urea product of type 2. Thisproduct can then be treated to deprotect the phosphate group or groups.

4. Glycerol Diamide Analogs

These compounds of the invention have an ester moiety attached to thecarbon which is beta to the phosphate group, instead of an amide moiety.

In general, these compounds are prepared by the etherification of aprotected chiral glycerol with an activated primary alcohol of a diolmoiety, followed by esterification of the secondary alcohol moiety andsubsequent deprotection of the glycerol moiety, to yield a new diol. Theprimary hydroxyl group of the diol is then protected, and the secondaryhydroxyl group is condensed with an acyl moiety to yield a diester. Theprimary hydroxyl is deprotected, followed by esterification with aphosphorylating agent, of which compound (11), below is exemplary.Following deprotection of the amine group introduced by thephosphorylating agent, the product is reacted with phosgene or adicarboxylic acid using a dehydrating agent such as EDC. Subsequentdeprotection of the phosphate groups yield compounds of the invention.

In the synthesis described generally above, the substituent at R¹ of thecompounds of the invention can easily be varied by utilizing differentdicarboxylic acid compounds. Such acids can be coupled to the aminegroup of the phosphate ester intermediate of the reaction schemeoutlined above, either using a dehydrating agent such as EDC, or byactivating the dicarboxylic acid by synthesizing, e.g., thecorresponding diacid chloride.

The substituents represented by variables R² and R⁵ in formula I abovecan easily be varied by utilizing an appropriate activated acid or acidchloride in the amidation or esterification reaction of the heteroatomrepresented by X or Y in formula I.

The substituents represented by variables R³ and R⁶ of formula I can bevaried by using an intermediate containing the desired number of carbonatoms which also contains an activated carbon functionality, e.g., ahalogen or sulfonate (OSO₂CH₃, OSO₂CF₃, OSO₂CH₂C₆H₄-p-CH₃) which can bereacted with the azido diol, amino alcohol, or glycerol startingmaterials.

The substituents represented by variables R⁴ and R⁷ in formula I abovecan be varied by using an appropriate activated acid or acid chloride inthe esterification of the secondary hydroxyl group used in the reactionschemes outlined above.

The values of a and b in compounds of formula I can be varied by usingthe appropriate compound (11) below. The values of variables d and e incompounds of formula I can be modified by using the appropriate2-aminodiol or 2-hydroxydiol starting materials

Adjuvant and Vaccine Formulation and Administration

The present invention is also directed to adjuvant formulationscomprising adjuvant compounds of the invention, as well as vaccine andother immunostimulatory formulations which comprise the adjuvantcompounds of the invention. Methods for the stimulation of an immuneresponse to a particular antigen are also within the scope of theinvention.

The host animals to which the adjuvant and adjuvant-containing vaccineformulations of the present invention are usefully administered includehuman as well as non-human mammals, fish, reptiles, etc.

Typically, an antigen is employed in mixture with the adjuvant compoundsof the invention. In other formulations of the adjuvant of the presentinvention, it may be useful in some applications to employ an antigencovalently linked to an amino, carboxyl, hydroxyl and/or phosphatemoiety of the adjuvant compounds of the invention. The specificformulation of therapeutically effective compositions of the presentinvention may thus be carried out in any suitable manner which willrender the adjuvant bioavailable, safe and effective in the subject towhom the formulation is administered.

The invention broadly contemplates therapeutic adjuvant formulations,which may for example comprise (i) at least one therapeuticallyeffective antigen or vaccine; and (ii) at least one adjuvant compoundaccording to the invention.

Such therapeutic composition may for example comprise at least oneantigenic agent selected from the group consisting of:

(A) live, heat killed, or chemically attenuated viruses, bacteria,mycoplasmas, fungi, and protozoa;

(B) fragments, extracts, subunits, metabolites and recombinantconstructs of (A);

(C) fragments, subunits, metabolites and recombinant constructs ofmammalian proteins and glycoproteins;

(D) tumor-specific antigens; and

(E) nucleic acid vaccines.

The therapeutic composition may therefore utilize any suitable antigenor vaccine component in combination with an adjuvant compound of theinvention, e.g., an antigenic agent selected from the group consistingof antigens from pathogenic and non-pathogenic organisms, viruses, andfungi, in combination with an adjuvant compound of the invention.

As a further example, such therapeutic compositions may suitablycomprise proteins, peptides, antigens and vaccines which arepharmacologically active for disease states and conditions such assmallpox, yellow fever, distemper, cholera, fowl pox, scarlet fever,diphtheria, tetanus, whooping cough, influenza, rabies, mumps, measles,foot and mouth disease, and poliomyelitis. In the resulting vaccineformulation, comprising (i) an antigen, and (ii) at least one adjuvantcompound of the invention the antigen and adjuvant compound are eachpresent in an amount effective to elicit an immune response when theformulation is administered to a host animal, embryo, or ovum vaccinatedtherewith.

In further embodiments, the compounds of the invention may be covalentlybonded to vaccine antigens, for example through an amino, carbonyl,hydroxyl or phosphate moiety. The compounds of the invention may bebonde, Methods of linking the adjuvant compositions of the invention tovaccine antigens are understood by persons of ordinary skill in the artin view of this disclosure. The adjuvant compositions may be linked tovaccines by any of the methods described in P. Hoffman et al., Biol.Chem. Hoppe-Sayler, 1989, 370:575-582; K.-H. Wiesmuller et al., Vaccine,1989, 7:29-33; K.-H Wiesmuller et al., Int. J. Peptide Protein Res.,1992, 40:255-260; J.-P. Defourt et al., Proc. Natl. Acad. Sci. 1992,89:3879-3883; T. Tohokuni et al., J. Am. Chem. Soc., 1994, 116:395-396;F. Reichel, Chem. Commun., 1997, 2087-2088; H. Kamitakahara, Angew.Chem. Int. Ed. 1998, 37:1524-1528; W. Dullenkopf et al., Chem. Eur. J.,1999, 5:2432-2438; all of which are hereby incorporated by reference.

The resulting vaccine formulations, including (i) an antigen, and (ii)an adjuvant compound, are usefully employed to induce an immunologicalresponse in an animal, by administering to such animal the vaccineformulation, in an amount sufficient to produce an antibody response insuch animal.

The modes of administration may comprise the use of any suitable meansand/or methods for delivering the adjuvant, adjuvant-containing vaccine,or adjuvant and/or antigen to one or more corporeal loci of the hostanimal where the adjuvant and associated antigens areimmumostimulatively effective. Delivery modes may include, withoutlimitation, parenteral administration methods, such as subcutaneous (SC)injection, transcutaneous, intranasal (IN), ophthalmic, transdermal,intramuscular (IM), intradermal (ID), intraperitoneal (IP),intravaginal, pulmonary, and rectal administration, as well asnon-parenteral, e.g., oral, administration.

The dose rate and suitable dosage forms for the adjuvant and vaccinecompositions of the present invention may be readily determined by thoseof ordinary skill in the art without undue experimentation, by use ofconventional antibody titer determination techniques and conventionalbioefficacy/biocompatibility protocols, and depending on the particularantigen or therapeutic agent employed with the adjuvant, the desiredtherapeutic effect, and the desired time span of bioactivity.

The adjuvant of the present invention may be usefully administered tothe host animal with any other suitable pharmacologically orphysiologically active agents, e.g., antigenic and/or other biologicallyactive substances.

Formulations of the invention can include additional components such assaline, oil, squalene, oil-water dispersions, liposomes, and otheradjuvants such as QS-21, muramyl peptides, Freunds's incompleteadjuvant, and the like.

SYNTHETIC EXAMPLES

All reaction products in the synthetic methods described below gavesatisfactory NMR spectra and thin layer chromatography profiles onsilica gel. All chromatography was performed on silica gel and theelution monitored by thin layer chromatography. All completed reactionswere determined by thin layer chromatographic analysis. All reactionswere run under nitrogen at room temperature unless otherwise specified.All reaction solvents were anhydrous unless otherwise noted. The typicalwork-up for the chemical reactions described below includes aqueouswashings, drying over anhydrous sodium sulfate and removal of solventunder reduced pressure.

Example 1 Succinate-1

To a solution of sodium azide (107.67 g) in 250 mL of water was added300 mL of methylene chloride. The mixture was cooled to 0° C. andtrifluoromethanesulfonic anhydride (57 mL) was added dropwise at a 0.32mL/minute rate. The mixture was stirred for an additional 6 hours at 0°C. and stored at −20° C. for 72 hours. The mixture was warmed to 10° C.followed by extraction with methylene chloride in a Teflon® separatoryfunnel. The combined organic layers were dried (magnesium sulfate). Theabove suspension was slowly filtered into a stirred solution of(±)-2-amino-1,3-dihydroxypropane (1) (9.89 g) in methanol (200 mL) and4-N,N-dimethylaminopyridine (DMAP, 54 g) at 10° C. The resultantreaction mixture was stirred for 17 hours at room temperature.

The solvent was removed under reduced pressure and the residue dissolvedin pyridine (200 mL) and cooled to 0° C. Acetic anhydride (50 mL) wasadded dropwise and the mixture stirred for 20 hours at room temperature.Additional acetic anhydride (20 mL) was added and after 4 hours, themixture was poured onto ice and worked up in the usual manner.Chromatography gave 16 g of diacetate (2) as an oil.

The diacetate (2) (16 g) was dissolved in methanol (150 mL) and sodiummetal (2.0 g) was slowly added. The mixture was stirred for 90 minutesand Dowex® 50-8 resin was added until the pH was less than or equal to7. The mixture was filtered followed by concentration of the filtrateand chromatography to give 6.73 g of the diol (3).

To a suspension of sodium hydride (1.24 g of a 60% oil dispersion washedthree times with hexanes and dried under nitrogen) in dimethylformamide(DMF, 200 mL) was added dropwise the azido-diol (3) (6.73 g) in THF (100mL), followed by the dropwise addition of3-R-hydroxy-1-O-tosyl-1-decanol (4) (tosylate, 9.44 g) in THF (100 mL).The mixture was stirred for 16 hours, diluted with methanol (200 mL),stirred with Amberlite® 25 H⁺ for 25 minutes and concentrated todryness. Chromatography gave 4.37 g of (5).

To a solution of the diol (5) (5.32 g) in methylene chloride (30 mL) wasadded triethylamine (TEA, 6 mL) and DMAP (trace), followed byt-butyldiphenylsilyl chloride (TBDPSC1, 5 mL) and the mixture wasstirred overnight. The mixture was worked up as usual. Chromatographygave 3.6 g of secondary alcohol (6) as an oil.

To a solution of the secondary alcohol (6) (2.95 g) in toluene (30 mL)was added pyridine (1.8 mL) followed by a slow addition of phosgene (4.5mL of a 1.93 M solution in toluene) at 0° C. After stirring at 0° C. for20 minutes, allyl alcohol (3.1 mL) was added dropwise. After anadditional stirring for 60 minutes at room temperature, the reaction wasworked up in the usual way. Chromatography gave 3.24 g of protectedalcohol (7) as an oil.

To a solution of protected alcohol (7) (1.29 g) in methylene chloride (3mL) was added hydrofluoric acid (HF, 4 mL) in acetonitrile (12 mL). Themixture was stirred overnight and worked up in the usual way.Chromatography gave 150 mg of the alcohol (8) as an oil.

To a solution of alcohol (8) (150 mg) in methylene chloride (0.6 mL) wasadded tetrazole (74 mg) and the phosphorylating reagent (11) (175 mg).After 30 minutes, oxone (323 mg) in a cooled THF (0.5 mL)-water (0.5 mL)solution was added to the cooled reaction mixture. After 3 hours, thereaction was worked up in the usual way. Chromatography gave 242 mg of(9) as an oil.

To make phosphorylating reagent 11, to a solution of distilleddiisopropylamine (9.0 mL) in methylene chloride was added tetrazole(4.51 g) at room temperature followed by stirring for 1.5 hours. Allylphosphorodiamidite (10) (20.5 mL) was added dropwise at a 6.5 mL/hourrate followed by stirring for an additional 3 hours.N-Boc-2-aminoethanol (10.36 g) in methylene chloride (50 mL) was addedto the above reaction mixture dropwise at a 8.4 mL/hour rate followed bystirring for an additional 18 hours. The white suspension was filteredthrough Celite 545 with two 20 mL washings with methylene chloride. Thefiltrate was concentrated followed by the suspension and filtering ofthe residue with hexanes (200 mL). The resulting hexanes filtrate wasconcentrated to dry and azeotroped with 2,10-mL portions of toluene toprovide the crude product (11) (21.54 g) as an oil.

To a suspension of dithiophenol tin (1.3 g) in methylene chloride (7.8mL) was added thiophenol (400 μL) followed by TEA (543 μL). The reactionmixture was stirred at room temperature for 15 minutes followed bystopping the stirring and allowing the residue to settle to the bottomof the flask. 1.0 mL of the above solution was added to a solution ofthe azide (9) (242 mg) in methylene chloride (0.5 mL) and allowed tostir for 30 minutes. Quenching with 0.1 N NaOH followed by the usualwork-up afforded 193.1 mg of the amine (12) as an oil.

To a dried solution of the amine (12) (193 mg) and acyl acid (which canbe made according to Christ et al., U.S. Pat. No. 5,530,113) (132 mg) inmethylene chloride was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 93mg). After stirring at room temperature for 90 minutes the reaction wasquenched and processed in the usual way to provide 232 mg of protectedphosphate (13) as an oil.

To a solution of the protected phosphate (13) (232 mg) in methylenechloride (1 mL) was added triethylsilane (TES, 120 μL) andtrifluoroacetic acid (TFA, 1.2 mL) followed by stirring for 30 minutes.The TFA was removed under reduced pressure followed by azeotroping with3, 5-mL portions of toluene. 20 mL of methylene chloride was added andthe mixture was worked up in the usual manner to give 174 mg of freeamine (14) as an oil.

To a dried solution of the free amine (14) (174 mg) in methylenechloride (0.5 mL) was added succinic acid (12.1 mg) and EDC (59 mg).After 1 hour, the reaction was worked up in the usual manner.Chromatography gave 143.1 mg of blocked diphosphate (15) as an oil.

To a solution of blocked diphosphate (15) (177.9 mg) in degassedchloroform (1.7 mL) in a dry box was first added phenylsilane (PhSiH₃,50 μL) and then tetrakistriphenylphosphine palladium (0) (Pd(PPH₃)₄, 70mg). After 1 hour, the reaction was removed from the box andchloroform:methanol:water, 2:3:1, was added and the mixture stirred for1 hour. It was poured onto a diethylamino-ethylcellulose (DEAE)chromatography column. Elution of the column with a linear gradient of0.0 M to 0.1 M ammonium acetate in chloroform:methanol:water, 2:3:1,extraction of the desired fractions with an equal volume of chloroform,concentration to dryness and the addition of 0.1 N NaOH (175 μL)followed by lyopholization gave 136.2 mg of (16) as a white solid.

Example 2 Chiral Malonate-Type 1

To a cooled solution of potassium carbonate (165 g) in water (575 mL)was added methylene chloride (200 mL) followed by ethyl benzimidatehydrochloride (17) (100 g) after which time the mixture was stirred for8 minutes. The layers were separated and the aqueous layer extractedwith methylene chloride. The organic layers were combined, dried and thesolvent removed under reduced pressure to give 83 g of (18).

To a solution of L-serine methyl ester hydrochloride (19) (41.6 g) in1,2-dichloroethane (450 mL) was added ethyl benzimidate (18) (36 g). Themixture was heated to reflux for 20 hours, cooled, filtered throughdiatomaceous earth, and concentrated to dryness to give 56 g of ethylester (21) as a white solid.

To an ice cold solution of ethyl ester (21) (56 g) in THF (500 mL) wasadded dropwise diisobutylaluminum hydride (DIBAL, 545 mL of a 1 Msolution in hexane). The mixture was allowed to warm to room temperatureovernight and then carefully poured onto an aqueous solution ofRochelle's salt (500 g in 1.0 L water). The mixture stirred for 1 hourand worked up in the usual manner. Chromatography gave 25.5 g of alcohol(22) as a white solid.

To a suspension of washed sodium hydride (5.3 g of a 60% oil dispersion)in DMF (200 mL) was added the alcohol (22) (24 g) in 250 mL of THF After30 minutes, the tosylate (4) was added in 250 mL of THF over 2.5 hoursand stirred overnight. The mixture was cooled in ice, methanol wasadded, the solvent removed under reduced pressure and chromatographed togive 4.32 g of alcohol (24) as an oil.

The alcohol (24) (4.3 g) was dissolved in 4 N aqueous hydrochloric acidand heated to reflux for 20 hours. The mixture was cooled, filtered,extracted with ether, made basic with sodium hydroxide and extractedtwice with chloroform. The combined chloroform layers were dried and thesolvent removed to give 2.88 g of diol (25) as an oil.

The diol (25) (2.88 g) was dissolved in saturated aqueous sodiumbicarbonate (45 mL) and THF (25 mL) and allowed to stir for fiveminutes. Myristoyl chloride (3.4 mL) was added dropwise over a 25-minuteperiod after which time the reaction mixture was allowed to stir for anadditional hour. The reaction was worked up in the usual manner andchromatographed to give 3.07 g of alcohol (26) as an oil.

To a solution of the alcohol (26) (1.78 g) in methylene chloride (140mL) was added tetrazole (683 mg), followed by the phosphorylatingreagent (11) (1.6 mL). After 30 minutes, the mixture was cooled in ice,and THF (105 mL) was added followed by an oxone solution (3 g in 90 mLof water). After 5 minutes, the ice bath was removed and the mixturestirred for 30 minutes. The reaction was worked up in the usual mannerand chromatographed to give 2.99 g of alcohol (27) as an oil.

To a solution of the alcohol (27) (3.9 g) in methylene chloride (126 mL)was added EDC (10.8 g), DMAP (66 mg) and dodecyl acid (1.62 g) andstirred overnight. Additional acid (1.6 g), EDC (1 g) and DMAP (0.5 g)was added. After 3 hours, the reaction was worked up in the usual mannerand chromatographed to give 2.07 g of N-BOC-protected amine (28).

To a solution of the N-BOC-protected amine (28) (187.3 mg) in methylenechloride (1.5 mL) was added TES (240 μL) and TFA (300 μL) and themixture stirred for 45 minutes. Toluene was added and the solventremoved under reduced pressure. The residue was dissolved in methylenechloride and worked up in the usual manner to give 154 mg of amine (29)as an oil.

To an ice cold solution of the amine (29) (75.6 mg) and malonic acid(4.8 mg) in methylene chloride (0.5 mL) was added EDC (27 mg), followedby removal of the cooling bath. After 1 hour, a trace of DMAP was added.After 2.5 hours, the mixture was directly chromatographed to give 54.1mg of dimer product (30).

To a solution of the dimer (30) (54 mg) in degassed chloroform (2 mL)was added PhSiH₃ (14 μL) and Pd(PPh₃)₄ (18 mg) followed by removal ofthe cooling bath. After 1 hour, the reaction was quenched withchloroform:methanol:water (2:3:1) and poured onto a DEAE cellulosechromatography column and chromatographed to give a semi-solid. Thesolid was dissolved in sterile water and 0.1 N aqueous sodium hydroxide(306 μL) was added and the mixture lyophilized to yield white solid (31)(25 mg).

Example 3 Chiral Malonate-Type 2

To a solution of D-serine methyl ester hydrochloride (32) (25 g) indichloroethane (270 mL) was added ethyl benzimidate (20) (24 g). After20 hours at reflux, the mixture was cooled to room temperature, filteredthrough diatomaceous earth and the solvent removed under reducedpressure to give 34 g of methyl ester (33) as a white solid.

To a ice cold solution of the methyl ester (33) (34 g) in THF (300 mL)was added dropwise a solution of DIBAL in hexane (1.0 M, 322 mL). Themixture was allowed to warm to room temperature overnight and thencarefully poured into an aqueous solution of Rochelle's salt. Themixture was then stirred for 1 hour and worked up. Chromatography gave18.6 g of alcohol (36) as a white solid.

To a suspension of washed sodium hydride (4 g of a 60% oil suspension)in DMF (100 mL) was added a solution of the alcohol (36) (8.6 g) in THF(40 mL). The mixture was stirred for 1 hour and a solution of thetosylate (23) (17.5 g) in THF (40 mL) was added. The mixture was stirredovernight and then additional tosylate (23) was added (5 g) and stirredfor another 4 hours. Methanol was added to the cooled reaction mixture,the solvent was removed under reduced pressure, and the residue waschromatographed to give 1.03 g of alcohol (37) as a solid.

The alcohol (37) (1.03) was dissolved in 4 N aqueous hydrochloric acid(25 mL) and the mixture was heated to 100° C. for 20 hours. Additionalhydrochloric acid (5 mL) was added and the reflux continued for 6 hours.The mixture was cooled, washed with ether, made basic with 1 N aqueoussodium hydroxide, and extracted (3×) with chloroform. The combinedchloroform layers were dried and the solvent removed under reducedpressure to give 553 mg of amino-diol (39).

To a solution of amino-diol (39) (553 mg) in THF (3 mL) was addedsaturated aqueous sodium bicarbonate (6 mL) followed by myristoylchloride (628 μL). After 50 minutes, the reaction was worked up in theusual way. Chromatography gave 389 mg of amide-diol (41) as an oil.

To a solution of the amide-diol (41) (531 mg) in methylene chloride (40mL) was added tetrazole (203 mg) and the mixture was stirred for 5minutes. Then phosphorylating reagent (11) (482 mg) was added. After 20minutes, additional phosphorylating reagent (11) (100 mg) was added andafter an additional 20 minutes, 100 mg more was added. After anadditional 20 minutes, 50 mg more was added. After 20 minutes, themixture was poured into a cold solution of THF (30 mL)/oxone (1.07g)/water (30 mL). The mixture was stirred at 0° C. for 5 minutes, andthen 20 minutes at room temperature after which time the reaction wasworked up in the usual manner. Chromatography gave 852 mg of phosphatealcohol (42) as an oil.

To a solution of the phosphate alcohol (42) (3.9 g) in methylenechloride (126 mL) was added EDC (10.8 g), DMAP (66 mg) and dodecyl acid(1.62 g). The reaction mixture was stirred overnight. Additional acid(1.6 g), EDC (1 g) and DMAP (0.5 g) was added. After 3 hours, thereaction was worked up in the usual manner and chromatographed to give2.07 g of protected amine (43).

To a solution of the protected amine (43) (194 mg) in methylene chloride(1.5 mL) was added TES (240 μL), and TFA (300 μL). The mixture wasstirred for 20 minutes and additional TES (50 μL) and TFA (50 μL) wasadded. After 1 hour, toluene was added and the solvent removed underreduced pressure and then the mixture was worked up in the usual manner.The crude free amine product (44) was used immediately in the nextreaction.

To an ice cold solution of the free amine (29) (43.5 mg) in methylenechloride (250 μL) was added mono-t-butyl malonate (8.3 μL), EDC (12.4mg) and a trace of DMAP. The ice bath was removed and after 2 hours, thereaction was worked up in the usual manner. Chromatography gave 44 mg ofamide (45).

To a solution of the amide (45) (44 mg) in methylene chloride (0.5 mL)was added TES (90 μL) and TFA (100 μL). After 2 hours, toluene was addedand the solvent removed under reduced pressure. The mixture was workedup in the usual manner to give 44.2 mg of free acid (46).

To an ice cold solution of the free acid (46) (41 mg) and the free amine(44) (26.3 mg) in methylene chloride (500 μL) was added EDC (13 mg) andthe mixture was stirred for 30 minutes. Additional EDC (5 mg) and DMAP(2 mg) was added and after 1 hour, the reaction was worked up in theusual manner. Chromatography gave 32.7 mg of coupled compound 47 as anoil.

To a solution of the coupled compound (47) (32.7 mg) in degassedchloroform (1.5 mL) in a dry box was added PhSiH₃ (8.5 μL) and Pd(PPH₃)₄(11 mg). The mixture was removed from the box and cooled in ice. After 5minutes, the ice bath was removed and after 1 hour,chloroform:methanol:water (2:3:1) was added and the mixture stirred for15 minutes and stored in the freezer overnight. The mixture was thenpoured onto a DEAE chromatography column. Chromatography gave 13.9 mg ofa compound (48) as a white powder after 0.1 N NaOH treatment followed bylyophilization.

Example 4 Chiral Urea-Type 1

To a solution of amine (44) (46.1 mg) in toluene was added saturatedsodium bicarbonate (0.5 mL) followed by phosgene (15 μL of a 1.93 Msolution in toluene). After 30 minutes, additional phosgene (10 μL) wasadded. After 2 hours, additional phosgene (5 μL) was added. The reactionwas quenched with aqueous sodium bicarbonate and worked up in the usualmanner to give 29.6 mg of urea (49) with protected phosphates.

To a solution of the urea with protected phosphates (49) (29.6 mg) indegassed chloroform (1.5 mL) in a dry box was added PhSiH₃ (8 μL). Thereaction vessel was removed from the dry box and placed on ice.Pd(PPh₃)₄ (10 mg) was added and after 5 minutes the ice bath removed.After 1 hour, the reaction was quenched by addition ofchloroform:methanol:water. The mixture was stirred for 15 minutes andstored overnight in the freezer. It was chromatographed on DEAE to give24.1 mg of (50) as a white powder after 0.1 N NaOH treatment followed bylyophilization.

Example 5 Chiral Urea-Type 2

To an ice-cold solution of trichloromethyl chloroformate (2.6 μL) inmethylene chloride (200 μL) was added a solution of free amine (29) (35mg) and 1,8-bis-(dimethylamino)-naphthalene in methylene chloride (200μL). After 5 minutes, the ice bath was removed. After 15 minutes,additional methylene chloride was added and the mixture worked up in theusual manner. Chromatography gave 9.4 mg of isocyanate (51) as an oil.

To a solution of the isocyanate (51) (9.4 mg) in methylene chloride (0.2mL) was added a solution of the amine (44) (10.3 mg) in methylenechloride. After 15 minutes, the reaction was worked up in the usualmanner. Chromatography gave 5.5 mg of coupled compound (61) as an oil.

To a solution of the coupled compound (61) (25.2 mg) in degassedchloroform (0.5 mL) in a dry box was added PhSiH₃ (6.6 μL) and Pd(PPH₃)₄(8.8 mg). The mixture was removed from the box and cooled in ice. After5 minutes, the ice bath was removed and after 1 hour,chloroform:methanol:water (2:3:1) was added and the mixture stiffed for15 minutes and stored in the freezer overnight. The mixture was thenpoured onto a DEAE chromatography column. Chromatography gave 7.5 mg of(62) as a white powder after 0.1 N NaOH treatment followed bylyophilization.

Example 6 Chiral Glycerol Analogue of Type 1

To a stirred suspension of sodium hydride (145.5 mg of 60% oildispersion washed with hexanes) in DMF (12 mL) was added (S)-(+)-alcohol(63) (0.41 mL) in 8 mL of THF dropwise over a 1 hour period. The mixturewas stirred for an additional 30 minutes followed by a dropwise additionof the tosylate (4) (0.789 g) in 10 mL of THF over a 10-minute period.The resulting reaction mixture was stirred overnight. The usual work upgave 0.56 mg of the desired adduct (65).

A solution of lauric acid (1.40 g), EDC (1.35 g), DMAP (0.04 g) and thealcohol adduct (65) (0.564 g) in 4 mL of methylene chloride was stirredfor 15 hours at room temperature. Brine and saturated aqueous sodiumbicarbonate (1:1) were added and the mixture extracted with methylenechloride. The mixture was worked up in the usual manner andchromatographed. The desired fraction was dissolved in 20 mL of 4:1acetic acid:water and stirred for 15 hours. The solvent was removedunder reduced pressure and the residue chromatographed to give 0.77 g ofsemi-solid diol (66).

A solution of the diol (66) (0.22 g), DMAP (6.3 mg), TEA (100 μL) andTBDPSC1 (164 μL) was stirred for 24 hours at room temperature. Methanol(2 mL) and a trace of aqueous hydrochloric acid was added followed byextraction with methylene chloride. The mixture was worked up in theusual way. The residue was chromatographed to give 0.3 g of alcohol (67)as an oil.

A solution of the alcohol (67) (0.3 g), DMAP (5.5 mg), EDC (258 mg),myristic acid (308 mg) in methylene chloride (4 mL) was stirred for 18hours at room temperature followed by the addition of brine andsaturated sodium bicarbonate. The mixture was worked up in the usual wayand chromatographed to give 0.4 g of silyl protected ether product (68).

To a solution of the silyl protected ether (68) (195 mg) in acetonitrile(2.7 mL) was added 48% hydrofluoric acid (0.756 ml). After 30 hours,saturated sodium bicarbonate was added and the mixture worked up in theusual way. Chromatography gave 94.7 mg of free alcohol (69).

To a solution of the free alcohol (69) (57 mg) in methylene chloride(0.5 mL) was added tetrazole (15.6 mg) and phosphorylating reagent (11)(40 mg) at room temperature. After four hours, the mixture was cooled to0° C. followed by the addition of oxone (82 mg) in THF (0.5 mL):water(0.6 mL). The mixture was warmed to room temperature and stirred for 80minutes. The final reaction mixture was worked up in the usual manner.Chromatography gave 72 mg of protected phosphate (70).

To a solution of the protected phosphate (70) (72 mg) in methylenechloride (1 mL) was added TES (120 μL) and trifluoroacetic acid (0.6 mL)followed by stirring for 1 hour. The TFA was removed under reducedpressure followed by azeotroping with 10 mL of toluene. 20 mL ofmethylene chloride was added and the mixture was worked up in the usualmanner to give 0.52 g of an oil.

The crude amine was dissolved in methylene chloride (0.7 mL) followed bythe addition of malonic acid (4.5 mg) and EDC (25.6 mg). The mixture wasstirred overnight and worked up in the usual way to give 32.5 mg of thedimer product (71).

The protected dimer (71) (32.5 mg) from the preceding reaction wasdissolved in degassed chloroform (2 mL) and PhSiH₃ (8.6 μL) was added ina dry box. The mixture was removed from the dry box and Pd(PPh₃)₄ (22.6mg), previously weighed in the dry box, was added. After 2 hours, themixture was chromatographed on DEAE to give 27.9 mg of white solid (72)after the addition of 1N sodium hydroxide (34.2 μL) followed bylyophilization.

Preparation of ER-804253

The alcohol (558 mg) was dissolved in methylene chloride (5 mL) cooledto 0° C. and triethylamine (0.466 mL) was added under a nitrogenatmosphere. After stirring for 5 minutes methanesulfonyl chloride (0.142mL) was added dropwise. The mixture was stirred for an additional 5minutes at 0° C. and then warmed to room temperature. After stirring foran additional hour, the mixture was worked up with sat. sodiumbicarbonate, extracted with ethyl acetate and the extract washed withwater, dilute aqueous hydrochloric acid, water, brine, dried and thesolvent removed to give 630 mg.

The mesylate (630 mg) and sodium azide (299 mg) were dissolved in DMSO(6 mL) and heated to 60° C. for 90 minutes. After cooling to roomtemperature the reaction mixture was diluted with methylene chloride,washed with water and brine. After extracting the aqueous washes thecombined organic was dried, concentrated, purified over silica gel usinga 4:1 ratio of hexanes to ethyl acetate and the dried product fractionsgive 420 mg.

The azide (295 mg) was dissolved in ethanol (5 mL) and Lindlar catalyst(200 mg) was added. After stirring under an atmosphere of hydrogen gasat atmospheric pressure, the filtered solution was dried to give 274 mg.

The amine (930 mg) was dissolved in THF (10 mL) and sat. sodiumbicarbonate (22 mL). After stirring for 5 minutes, lauroyl chloride(0.712 mL) was added dropwise over 20 minutes. The final mixture wasextracted with chloroform, dried to give 1.45 g.

The amide (1.11 g) was dissolved in methanol (14 mL) and 4 Nhydrochloric acid (8 mL) added. The mixture was stirred for 1 our at 50°C. and then concentrated. Methanol (16 mL) and 40% sodium hydroxide (8mL) was added and the mixture refluxed for 1 hour. It was cooled,extracted with methylene chloride and the extract washed with water,dried and the solvent removed to give 930 mg.

The amino alcohol was dissolved in THF (6 mL) and saturated sodiumbicarbonate added (13 mL). After 5 minutes the mixture was cooled to 0°C. and myristoyl chloride (300 μL) added. After 30 minutes, the mixturewas worked up in the usual way to give 430 mg.

The alcohol (101.7 mg) was dissolved in ice cold methylene chloride andphosphorylating reagent 11 (90 μL) was added and the mixture stirred for30 minutes. Ice cold oxone (166.3 mg) was added and the mixture stirredfor 30 minutes. The reaction was quenched with thiosulfate. The mixturewas worked up the usual way and chromatographed to give 174 mg (notpurified)

The protected amine from the above reaction was dissolved in ice coldmethylene chloride (1 mL), trifluoroacetic acid (1 mL) was added and themixture stirred for 1 hour. The TFA was removed and the mixture purifiedto give 106.7 mg.

The amine was dissolved in methylene chloride (3 mL) and saturated.sodium bicarbonate solution (3 mL) was added. The mixture was cooled inice and phosgene in toluene (0.55 equiv.) was added dropwise. Themixture was stirred for 20 minutes and worked up to give 112.3 mg.

To a solution of the blocked phosphate (40.5 mg) in ice cold chloroform(2.6 mL) was added phenylsilane (10.7 mg) andtetrakis(triphenylphosphine)palladium [0] (28.7 mg) and the mixturestirred for 1 hour. The mixture was chromatographed on a DEAE column togive 27.7 mg of ER-804253.

Preparation of ER-804130

To a solution of the amide (3 g) in THF (65 mL) at −78° C. was added anequivalent of butyllithium, followed by a solution of nonanoyl chloridein THF (6 mL). Aqueous ammonium chloride was added and the mixtureworked up in the usual manner to give 5.35 g.

To a solution of the alcohol (5 g) in ice cold methylene chloride (100mL) was added triethylamine (4.1 mL) and mesyl chloride (2.1 mL). Themixture was stirred for 4 hours and worked up in the usual manner togive 6.99 g.

To the solution of the mesylate (6.99 g) in ice cold DMF (100 mL) wasadded potassium bromide. The mixture was allowed to warm to roomtemperature and stirred for five hours. It was worked up in the usualmanner to give 4.63 g of clear oil.

A solution of the amide (2.8 g) in THF (15 mL) was added to a −78° C.solution of sodium bis-trimethylsilylamide in THF (15 mL). After 1 hour,the bromide was added and the mixture allowed to warm to roomtemperature and worked up in the usual manner to give 1.02 g.

To a solution of the olefin (1.02 g) in EtOAc was added palladium oncarbon (126 mg) and the mixture placed under hydrogen. After 4 hours,the mixture was worked up in the usual manner to give 1.0 g.

To a solution of the amide (1.0 g) in ice cold THF (20 mL) was addedwater, hydrogen peroxide and lithium hydroxide. The next day, themixture was worked up in the usual manner to give 590 mg of acid.

To a solution of the acid in ice cold THF (10 mL) was added diborane:THFcomplex and the mixture allowed to warm slowly. After seven hours,dilute hydrochloric acid was added carefully and the mixture worked upin the usual manner. The crude material was dissolved in ice cold etherand LAH solution (2 mL, of 1 M) added. After 5 minutes, the mixture wasworked up in the usual manner to give 556 mg of the alcohol.

To a −78° C. solution of oxalyl chloride (2.2 mL) in methylene chloride(10 mL) was added DMSO (1.1 mL) and after 2 minutes the alcohol (556 mg)was added in methylene chloride (5 mL). After 20 minutes, triethylamine(1 mL) was added and the mixture warmed to 0° C. The mixture was dilutedwith ether and worked up in the usual manner to give 567 mg.

The Wittig reagent (679 mg) was suspended in THF (10 mL) and KHMDSsolution (4 mL of 0.5 M) added. After 20 minutes, the mixture was cooledto −78° C. and the aldehyde (567 mg) in THF (5 mL) was added. After 15minutes, the mixture was worked up in the usual manner to give 342 mg.

To a solution of the enol ether (342 mg) in acetonitrile (3.5 mL) andwater (0.15 mL) was added hydroiodic acid. After 4 hours, the mixturewas worked up in the usual manner to give 325 mg.

To a solution of the aldehyde (325 mg) in methanol (10 mL) was addedsodium borohydride (38 mg). After 3 hours, the reaction was worked up inthe usual manner to give 303 mg of the alcohol.

To an ice cold solution of the alcohol (303 mg) in methylene chloride(10 mL) was added triethylamine (150 μL) and mesyl chloride (76 μL).After 4 hours, the reaction was worked up in the usual manner to give352 mg.

To a solution of the oxazoline (1 mL) in ice cold THF (5 mL) was addedpotassium t-butoxide solution (2.2 mL of 1M). After 30 minutes, themesylate was added in THF (5 mL) and the mixture stirred for 8 hours.The usual work-up gave 318.5 mg.

A solution of the oxazoline in methanol (8 mL) and hydrochloric acid (4mL of 4M) was warmed to 50° C. for 90 minutes. Additional methanol wasadded and the solvent removed. The residue was dissolved in methanol (8mL) and sodium hydroxide solution (4 mL) and briefly warmed to 50° C.The mixture was cooled and extracted with chloroform. The usual work-upgave 114 mg.

To a solution of the amine (114 mg) in THF (2 mL) and saturated aqueoussodium bicarbonate (2 mL) was added the acid chloride. After 30 minutesadditional acid chloride was added. After 30 minutes, the reaction wasworked up in the usual manner to give 146 mg.

To a solution of tetrazole (48 mg), the phosphorylating reagent (122 mg)in ice cold methylene chloride (2 mL) was added the alcohol (146 mg).Oxone (230 mg) in water (1 mL) and THF (2 mL) was added. After 90minutes, thiosulfate was used to quench the reaction. Standard work upgave 140 mg.

To a solution of the substrate in ice cold methylene chloride was addedtriethylsilane (370 μL) and trifluoroacetic acid (110 μL). After 5minutes, the volatiles were removed to give 148 mg.

To a solution of the amine (66.9 mg) in ice cold methylene chloride (0.8mL) was added saturated aqueous sodium bicarbonate (0.8 mL) and phosgenesolution (20 μL of 1.93 M). After one hour, additional phosgene (10 μL)was added. After 30 minutes, the usual work-up gave 47.7 mg.

To a solution of phenylsilane (15 μL), tetrakis(triphenylphosphine)palladium [0] (24.8 mg) in ice cold chloroform under an inert atmospherewas added the substrate. After 5 minutes, the mixture was applied to aDEAE column and chromatographed to give 31 mg of ER-804130.

Preparation of ER-804558

To a solution of the amine (325 mg) in methylene chloride was addedtriethylamine (321 μL) and 1-dodecanesulfonyl chloride. After 3 hours,the usual work up gave 384 mg.

To a solution of the protected alcohol (384 mg) in THF (4 mL) was addedtetrabutylammonium fluoride (123 mg) and acetic acid (29 μL). After 2hours, the usual work up gave 180 mg.

The remainder of the synthesis was completed as outlined above for othercompounds of the present invention, i.e. phosphorylating, deblocking,coupling with phosgene, and deprotecting with phenylsilane andpalladium.

Preparation of ER-804442

The diol amine was mono-protected as its t-butyl-diphenylsilyletheroutlined above.

The amine (2.6 g) and benzophenone imine (1.1 mL) were mixed and heatedto 40° C. for 4 days to give after chromatography 3.3 g.

To a solution of the imine (3.3 g) was in ice cold methylene chloridewas added lauric acid (1.5 g), EDC (1.7 g) and DMAP (155 mg). The nextday, the reaction was worked up in the usual manner to give 3.15 g.

To a solution of the imine (3.14 g) in ether was added 1 N aqueoushydrochloric acid. The next day, the reaction was worked up in the usualmanner to give 2.81 g.

To a solution of trichloromethylchloroformate (12 μL) in ice coldmethylene chloride (250 μL) was added dodecylamine (18 μL) anddiisopropylethylamine (27 μL). After 30 minutes, the solvent wasremoved. The residue was dissolved in ice cold methylene chloride, towhich was added the amine (55.6 mg) and additional diisopropylethylamine(13 μL). After 2 hours, the usual work up gave, after chromatography,60.9 mg.

This product was de-protected with fluoride, phosphorylated,de-protected with TFA, dimerized with phosgene, and un-blocked withphenylsilane and palladium as outlined above to give ER-804442.

Preparation of ER-804221

To an ice cold solution of glycine (8.26 g) in aqueous sodium hydroxide(4.4 g in 60 mL) was added lauroyl chloride (21.8 g). After 1 hour, acidwas added and the mixture worked up in the usual way. Recrystallizationfrom ethyl acetate gave 9.7 g.

To an ice cold solution of the amine (1.4 g) in methanol was addedtriflic azide (20 mg). The next day, additional azide was added. After 2hours, the reaction was worked up in the usual manner to give afterchromatography 1.14 g.

To a solution of the alcohol (1.14 g) in methylene chloride was addedt-butyl-diphenylsilyl chloride (1.09 mL), triethylamine (1.8 mL) andDMAP (50 mg). After 3 hours, the usual work up gave 1.4 g.

To a solution of the alcohol (1.4 g) in ice cold methylene chloride wasadded lauric acid (826 mg), EDC (1.05 g) and DMAP (33 mg). The next day,the usual work up gave after chromatography 778 mg.

To a solution of the azide (778 mg) in THF was added acetic acid (77 μL)and TBAF (323 mg). The next day, the usual work up gave, afterchromatography, 428 mg.

To a solution of the azide (460 mg) in methylene chloride was addedtetrazole (165 mg), the phosphorylating reagent (390 mg), and after 30minutes, oxone in water (722 mg in 3 mL). The reaction was quenched withthiosulfate. Usual work up, after chromatography, gave 392 mg.

The protected amine (460 mg) was dissolved in methylene chloride andtrifluoroacetic acid (394 μL) and triethylsilane (308 μL). After 1.5hour, the usual work up gave, 392 mg.

To an ice cold solution of the amine in methylene chloride (5.5 mL) wasadded saturated sodium bicarbonate (5.5 mL), and phosgene (164 μL of a1.93 M solution in toluene). After 15 minutes, the usual work up, afterchromatography gave 342 mg.

To a ice cold solution of the azide (187 mg) in methylene chloride wasadded the tin reagent (1.5 mL) which was prepared as outlined in U.S.Pat. No. 5,756,718 incorporated herein by reference. After 30 minutes,the mixture was chromatographed to give 187 mg.

To a ice cold solution of the urea (55 mg) in methylene chloride wasadded the acid (59 mg) (prepared as above) and EDC (44 mg). The nextday, additional EDC (5 mg) and acid (5 mg) was added. After 2 hours, thenormal work up provided 45.7 mg. Normal removal of the protecting groupswith phenylsilane and palladium gave ER-804221.

ER-804222 was prepared in a similar manner except that the condensationproduct between lauryl chloride and glycine, 15-methylmyristic acid wasused.

Preparation of ER-804281

To a ice cold solution of the protected alcohol (8.3 g) inacetonitrile:water was added CAN (41.4 g). After 1 hour, the usual workup gave 5.7 g.

A solution of the alcohol (5.63 g) in 4 N HCl solution was heated toreflux for 1 hour, cooled, neutralized with sodium hydroxide and workedup in the usual manner to give 2.1 g.

To an ice cold solution of the alcohol (2.2 g) in rmiethylene chloridewas added imidazole (0.7 g), t-butyl-diphenylsilyl chloride in 15 mL ofmethylene chloride. The next day, the usual work up gave 1.54 g.

To a solution of the alcohol (1.93 g) in methylene chloride (40 mL) wasadded benzophenone imine (0.8 mL). After 1 day, the mixture was heatedto reflux overnight. The usual work up gave 1.67 g.

To an ice cold solution of the alcohol (1.67 g) in methylene chloridewas added DMAP (159 mg), EDC (0.99 g) and lauric acid (1.04 g). Afterone day, the usual work up gave 74% yield.

To an ice cold solution of the imine (2.9 g) in ether (50 mL) was added1 N HCl (50 mL). The next day, the usual work up gave 2.09 g.

To a solution of the amine (1.24 g) in dichloroethane was added sodiumcyanoborohydride (178 mg) and tetradecanal (411 mg). The next day, theusual work up gave 1.5 g.

To a ice cold solution of the amine (221 mg) in dioxane was added allylchloroformate (40 mg) and 308 μL of 1 N NaOH solution. After 2 hours,the usual work up gave 200 mg.

To an ice cold solution of the protected alcohol (365 mg) in THF wasadded TBAF (1924 μL) and acetic acid (122 μL). The next day, the usualwork up gave 271 mg.

This material was phosphorylated, deblocked with TFA, dimerized withphosgene and the allyl protecting groups removed with phenylsilane andpalladium as described above to give ER-804281.

Preparation of ER-804339

ER-804281→ER-804339

To a ice cold solution of ER-804281 (7 mg) in methylene chloride wasadded triethylamine (5 μL), DMAP (0.6 mg) and acetyl chloride (1.8 μL).After 4 days, the usual work-up gave 1.1 mg.

Preparation of ER-804674

ER-804281→ER-804674

To a solution of ER-804281 (12.7 mg) in THF (1.0 mL) was added methyliodide (9.2 mg) and sodium bicarbonate (6.8 mg). The mixture was stirredfor 5 days and sodium bicarbonate (14 mg) and additional methyl iodide(8 mL) was added. After an additional 3 days, additional bicarbonate (28mg) and Mei (16 μL). After an additional 6 days, the mixture was workedup to give 9.1 mg of product.

Preparation of ER-804596

To a solution of the alcohol (393 mg) in methylene chloride (2 mL) wasadded diisopropylamine (210 μL), tetrazole (105 mg) and phosphorylatingreagent (as described above) (488 mg). After 2⅕ hours, the usual work upgave the desired product.

To a solution of the diol (73 mg) in acetonitrile was added tetrazole(175 mg), the azide (1 equivalent). After 3 hours, the mixture wascooled and ozone (1229 mg) added. The next day, usual work up gave thedesired product.

To an ice cold solution of the protected alcohol (92.9 mg) inacetonitrile:water (6 mL:1.5 mL) was added CAN(358 mg). After 1 hour,the usual work up provided 68.5 mg.

To an ice cold solution of the diol (68.5 mg) in methylene chloride wasadded lauric acid (76.5 mg), DMAP (4.7 mg) and EDC (73 mg). The nextday, the usual work up gave 76.5 mg.

The azides were reduced using the tin reagent described above. Thediamine was acylated with dodecanoyl chloride, and the protecting groupsremoved with phenylsilane and palladium as described above to giveER-804596.

Preparation of ER-804732

The alcohol (7.04 g) was dissolved in methylene chloride (300 mL) withtriethylamine (11.13 mL) and then cooled to 0° C. under a nitrogenatmosphere. Methanesulfonyl chloride (3.69 mL) was added dropwise afterwhich time the reaction was stirred at room temperature for 1 hour. Theusual work up gave 5.551 g.

The mesylated (1.114 g) was dissolved in DMF (30 mL) followed by sodiumazide (0.9337 g). The reaction mixture was warmed to 57° C. and stirredfor 16 hours and then to 104° C. for and additional 3 hours. Aftercooling to room temperature the mixture was worked up in the usualmanner and gave 0.466 g.

The protected aminoalcohol (0.466 g) was hydrolyzed using 4 N HCl (15mL) at 107° C. for 3 hours. After cooling to room temperature, thereaction mixture was filtered and extracted with ethyl ether, dried,concentrated and used in the next reaction.

The crude aminoalcohol was dissolved in THF (5 mL) with saturated sodiumbicarbonate (6 mL) and cooled to 0° C. Myristoyl chloride (0.79 mL) wasadded dropwise after which time the reaction was warmed to roomtemperature and stirred for 2 hours. The reaction mixture was worked upusing the usual methods and gave 0.751 g.

The alcohol (0.185 g) was dissolved in DMF (3.0 mL) with imidazole(0.077 g) and tert-butyldiphenylsilyl chloride (0.197 mL). The reactionmixture was stirred at room temperature for 16 hours after which timethe usual work up gave 0.320 g.

The azide (0.975 g) was dissolved in methanol (20 mL) with 10% palladiumon carbon (0.180 g). The mixture was stirred under an atmosphere ofhydrogen gas under atmospheric pressure for 2 hours after which time thegas was evacuated and the mixture filtered over Celite 545 andconcentrated. Purification using the usual methods gave 0.873 g.

DMSO (1.5 mL) was added dropwise to oxalyl chloride (0.92 mL) inmethylene chloride (30 mL) at 78° C. After stirring for 15 minutes thealcohol (1.727 g) in methylene chloride (30 mL) was added dropwise andstirred for an additional 30 minutes. Triethylamine (4.90 mL) was addeddropwise, the reaction was warmed to 0° C. and quenched using saturatedammonium chloride. Purification of the crude product using silica gelchromatography with 20% ethyl acetate in hexanes gave 1.653 g.

The primary amine (0.135 g) and aldehyde (0.077 g) were dissolved in1,2-dichloroethane (5 mL) followed by the addition of sodiumcyanoborohydride (0.032 g). The reaction was stirred for 20 hours afterwhich time acetic acid (0.02 mL) was added and the reaction worked up inthe usual manner to give 0.103 g.

The secondary amine was dissolved in 1,4-dioxane (15 mL) and cooled to0° C. followed by the slow addition of 1 M sodium hydroxide (3.0 mL).After stirring for 10 minutes allyl chloroformate (0.236 mL) was addeddropwise after which time the reaction was warmed to room temperatureand stirred for 16 hours. Work up in the usual manner gave 0.613 g.

The para-methoxybenzyl ether (0.613 g) was dissolved in a 4 to 1 ratioof acetonitrile to water (15 mL), cooled to 0° C. and then CAN (1.525 g)was added. The reaction mixture was stirred at 0° C. for 2 hours andthen worked up in the usual manner to give 0.357 g.

The alcohol (0.357 g) was dissolved in methylene chloride (5 mL) withlauric acid (0.184 g), EDC (0.175 g) and cooled to 0° C.4-Dimethylaminopyridine (0.012 g) was added and the resulting mixturewas stirred at room temperature for 2 hours. Work up in the usual mannergave 0.436 g.

The silyl protected alcohol (0.211 g) was dissolved in THF (5 mL) withacetic acid (0.03 mL). Tetrabutylammonium fluoride (0.115 g) was addedin one portion and the reaction mixture was stirred at room temperaturefor 16 hours. A normal work up gave 0.150 g.

This material was phosphorylated, deblocked with TFA, dimerized withphosgene and the allyl protecting groups removed with phenylsilane andpalladium as descried above to give ER-804732.

Preparation of ER-804680

The aldehyde (1.54 g) was dissolved in THF (28 mL) and cooled to 0° C.after which time 2-methyl-2-butene (14 mL) and tert-butyl alcohol (28mL) was added. A stirred suspension of sodium chlorite (3.70 g) andsodium trihydrogen phosphate (4.09 g) in water (42.7 mL) was added tothe above mixture and stirred at 0° C. for 1.5 hours. The completedreaction was diluted with ethyl acetate (100 mL) and washed with 10%sodium bisulfite, brine, dried, concentrated and silica gelchromatographed to give 1.55 g.

The amine (0.553 g) and acid (0.381 g) were mixed in methylene chloride(8 mL) and cooled to 0° C. after which time EDC (0.230 g) was added andthe reaction mixture was stirred at room temperature for 72 hours. Theusual work up gave 0.567 g.

The methoxybenzyl ether (0.567 g) was dissolved in a 1 to 1 ratio ofacetonitrile to water (16 mL) with methylene chloride (8 mL) and cooledto 0° C. CAN (1.53 g) was added and the reaction mixture was stirred for1 hour after which time it was worked up in the usual manner to give thecrude alcohol.

The crude alcohol from above was dissolved in methylene chloride (15 mL)with lauric acid (0.280 g) and 4 dimethylaminopyridine (0.017 g). Thereaction mixture was cooled to 0° C. and EDC (0.267 g) was added in oneportion after which time the reaction mixture was warmed to roomtemperature and stirred for 16 hours. Normal work up procedures provided0.622 g.

The silyl ether (0.563 g) was dissolved in THF (10 mL) with acetic acid(0.087 mL). Tert-butylammonium fluoride (0.330 g) was added and thereaction was stirred at room temperature for 16 hours. Work up in theusual manner gave 0.384 g.

This material was phosphorylated, deblocked with TFA, dimerized withphosgene and the allyl protecting groups removed with phenylsilane andpalladium as described above to give ER-804780.

Preparation of ER-804679

The protected secondary amine (0.071 g) was dissolved in degassedchloroform (3 mL) with phenylsilane (0.017 mL) and acetic anhydride(0.014 mL). The reaction mixture was cooled to 0° C. followed by theaddition of tetrakistriphenylphosphine palladium (0) (0.002 g). Thereaction mixture was warmed to room temperature and allowed to stir for30 minutes. The completed reaction was diluted with methylene chloride,washed with water, dried, concentrated, and chromatographed to give0.068 g.

The silyl ether was deprotected in THF (5 mL) with acetic acid (0.025mL) with the addition of tert-butylammonium fluoride (0.092 g). Afterstirring at room temperature for 16 hours the reaction was worked up inthe usual manner to give 0.120 g.

This material was phosphorylated, deblocked with TFA, dimerized withphosgene and the allyl protecting groups removed with phenylsilane andpalladium as described above to give ER-804679.

Preparation of ER-804764

DMSO (0.33 mL) was added dropwise to oxalyl chloride (0.203 mL) inmethylene chloride (10 mL) at 78° C. After stirring for 15 minutes thealcohol (0.993 g) in methylene chloride (3 mL) was added dropwise andstirred for an additional 30 minutes. Triethylamine (1.08 mL) was addeddropwise, the reaction was warmed to 0° C. and quenched using saturatedammonium chloride. Purification of the crude product using silica gelchromatography with 20% ethyl acetate in hexanes gave 0.743 g.

The 1.6 M n-butyl lithium in hexanes (1.5 mL) was added dropwise to thephosphonium salt (0.797 g) in THF (10 mL) at 0° C. After stirring for 30minutes the aldehyde (0.734 g) in THF (15 mL) was added dropwise. Afterstirring at room temperature for one hour the reaction was worked up inthe usual manner to give 0.193 g.

The enol ether (0.193 g) was hydrolyzed with 57% hydrogen iodide (0.114L) in acetonitrile (2 mL). After stirring at room temperature for 2hours the reaction was quenched with saturated sodium bicarbonate,extracted with methylene chloride, and dried to give 0.211 g crudealdehyde.

The crude aldehyde (0.211 g) was dissolved in methanol (3 mL) and sodiumborohydride (0.033 g) was added at 0° C. After stirring for 30 minutesthe reaction was diluted with water, extracted with methylene chloride,dried, concentrated and purified by silica gel chromatography to give0.148 g.

This material was phosphorylated, deblocked with TFA, dimerized withphosgene and the allyl protecting groups removed with phenylsilane andpalladium as described above to give ER-804764.

Preparation of ER-804772

The commercially available diol (1.486 g) was mixed with the acetal(1.864 g) and para-toluenesulfonic acid (0.195 g) in DMF (10 mL). Afterstirring for 20 hours at room temperature under a nitrogen atmosphere,the reaction was quenched with sat. sodium bicarbonate, extracted withmethylene chloride, dried and concentrated via high vacuum. Silica gelchromatography of the resultant crude product using 10% ethyl acetate inhexanes gave 2.084 g.

The acetal (2.084 g) was cooled to 78° C. under a nitrogen atmosphere inmethylene chloride (30 mL) followed by the dropwise addition of 1.O MDIBAL in hexanes (14.3 mL). After additional DIBAL (14 mL) was added,the reaction mixture was stirred for 1 hour, warmed to room temperatureand quenched with sodium, potassium tartarate. The normal work up gave2.1 g.

The alcohol (1.286 g) was mixed with triethylamine (0.883 g) inmethylene chloride (15 mL) and cooled to 0° C. Methanesulfonyl chloride(0.575 g) was added dropwise followed by stirring for 20 minutes at O°C. and room temperature for 2 hours. The normal work up gave 1.496 g.

The alcohol (1.495 g) in DMF (10 mL) was added dropwise to a stirringsuspension of washed 60% sodium hydride (0.257 g) in DMF (20 mL) at O°C. After stirring for 3 hours the mesylate (0.925 g) in DMF (10 mL) wasadded dropwise. After stirring for an additional 3 days, the reactionwas quenched and worked up in the usual manner gave 0.905 g.

As with examples provided above, the para-methoxybenzyl protecting groupwas hydrolyzed with CAN, the protected amino alcohol hydrolyzed usingaqueous HCl then KOH, acylation of the amine with tetradecanoylchloride, silylation of the primary alcohol with TBDPS, acylation of thesecondary alcohol with dodecanoyl chloride, and hydrolysis of the silylprotecting group using TBAF to give the primary alcohol. This materialwas phosphorylated, deblocked with TFA, dimerized with phosgene and theallyl protecting groups removed with phenylsilane and palladium asdescribed above to give ER-804772.

Preparation of ER-804947

The alcohol (0.263 g) in THF (5 mL) was added dropwise to washed 60%sodium hydride (0.216 g) in DMF (2.0 mL) at room temperature under anitrogen atmosphere. The reaction mixture was stirred for 30 minutesafter which time benzyl bromide (0.272 mL) with a catalytic amount (0.05g) of tetrabutylammonium iodide. The final reaction mixture was stirredfor an additional hour after which time the mixture was quenched andworked up in the usual manner to give 0.365 g.

The protected aminoalcohol (0.189 g) was hydrolyzed using 4 Nhydrochloric acid (2.5 mL) followed by 40% sodium hydroxide (2.5 mL) asdescribed previously to provide 0.121 g.

The aminoalcohol (0.121 g) was dissolved in methylene chloride (2 mL)with saturated sodium bicarbonate (2 mL). After cooling to 0° C.,myristoyl chloride (0.199 mL) was added dropwise. After continuedstirring for 2 hours the mixture was worked up in the usual manner andgave 0.181 g.

The alcohol (0.181 g) was dissolved in methylene chloride (5 mL) withthe acid (0.180 g) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide(EDC 0.133 g). The mixture was cooled to 0° C. and4-dimethylaminopyridine was added follow by stirring for 16 hours atroom temperature. The usual work up gave 0.310 g.

The para-methoxybenzyl ether (0.305 g) was dissolved in acetonitrile (8mL) with water (2 mL) and cooled to 0° C. Cerium ammonium nitrate (1.110g) was added and the reaction mixture was stirred for 2 hours afterwhich time using the normal work up gave crude alcohol.

The crude alcohol was dissolved in methylene chloride (8 mL) with lauricacid (0.126 g) and 4-dimethylaminopyridine (0.011 g). After cooling to0° C., EDC (0.119 g) was added and the mixture was stirred at roomtemperature for 16 hours. The usual work up gave 0.355 g.

The benzyl ether (0.355 g) was dissolved in ethyl acetate (50 mL) withpalladium hydroxide (0.048 g) and acetic acid (0.25 mL). The reactionmixture was placed under 50 psi of a hydrogen atmosphere and shaken for10 hours. Work up in the usual manner gave 0.255 g. This material wasphosphorylated, deblocked with TFA, dimerized with phosgene and theallyl protecting groups removed with phenylsilane and palladium asdescribed above to give ER-804947.

Preparation of ER 805718

To a stirred solution of diol (6.90 g) in methylene chloride (100 mL) atroom temperature was added triethylamine (6.63 mL) and DMAP (0.50 g)followed by the drop wise addition of tert-butyldiphenylchlorosilane(10.38 mL). After stirring for 18 h the reaction was worked up in thenormal fashion followed by silica gel purification to provide 11.70 g ofthe desired silyl ether.

To a stirred solution of silyl ether (11.70 g) in methylene chloride (60mL) at room temperature was added lauric acid (8.60 g). After themixture became a clear solution the reaction mixture was cooled to 0° C.followed by the addition of EDC (8.3 g) and DMAP (0.35 g). Afterstirring for 16 h the reaction was worked up in the normal fashion andpurified using silica gel chromatography to provide 16.43 g of thedesired ester.

To a stirred solution of ester (16.40 g) in THF (100 mL) at roomtemperature was added acetic acid (2.20 mL) followed by the drop wiseaddition of 1 M TBAF in THF (33 mL). After stirring for 16 h thereaction was worked up in the normal fashion and purified using silicagel chromatography to provide 10.32 g of the desired alcohol.

To a stirred solution of alcohol (1.03 g) in THF (12 mL) at 0° C. wasadded triphenylphosphine (1.50 g) and diphenylphosphoryl azide (1.24 mL)followed by a drop wise addition of DEAD (1.1 mL). After stirring for1.5 h the reaction was worked up in the normal fashion and purifiedusing silica gel chromatography to provide 0.95 g of the desired azide.

To a stirred solution of azide (0.473 g) in methanol (10 mL) at roomtemperature under an argon atmosphere was added 10% palladium on carbon(0.20 g) followed by charging the reaction vessel with hydrogen gas.After stirring at atmospheric pressure for 2 h the reaction was workedup in the normal fashion to provide 0.390 g of the desired amine.

To a stirred solution of amine (0.188 g) and protected L-serine (0.150g) in methylene chloride (2 mL) at 0° C. was added EDC (0.152 g)followed by DMAP (0.006 g). After stirring for 16 h the reaction wasworked up in the normal fashion and purified using silica gelchromatography to provide 0.292 g of the desired amide.

To a stirred solution of amide (0.292 g) in methanol (7 mL) at roomtemperature was added para-toluenesulfonic acid (0.70 g). After stirringfor 4 h the reaction was worked up using sat. sodium bicarbonatefollowed by extraction and concentration in the normal fashion. To astirred solution of the crude intermediate in methylene chloride (7 mL)was added at room temperature triethylsilane (1.2 mL) followed bytrifluoroacetic acid (2.1 mL). After stirring for 1 h the reactionmixture was worked up in the normal fashion and purified using silicagel chromatography to provide 0.170 g of the desired amino alcohol.

To a stirred solution of amino alcohol (0.170 g) in THF (4 mL) at roomtemperature was added sat. sodium bicarbonate (4 mL) followed bymyristoyl chloride (0.114 mL). After stirring for 3 h the reactionmixture was worked up in the normal fashion and purified using silicagel chromatography to provide 0.244 g of the desired alcohol.

To a stirred solution of alcohol (0.256 g) in methylene chloride (4 mL)at room temperature was added tetrazole (0.069 g) followed by thephosphorylating reagent (0.163 g). After stirring for 30 minutes atetrazole (0.065 g) and phosphorylating reagent (0.165 g) were added.The reaction mixture was stirred for an additional 1 h the reactionmixture and then poured over a stirred suspension of oxone (0.601 g) inTHF (4 mL) and water (4 mL) at 0° C. After stirring for an additional 16hours at room temperature, the reaction was worked up in the normalfashion and purified using silica gel chromatography to provide 0.40 gof the desired protected amino-phosphate.

To a stirred solution of the protected amino-phosphate (0.400 g) inmethylene chloride (4 mL) was added at room temperature triethylsilane(0.21 mL) followed by trifluoroacetic acid (0.34 mL). After stirring for2 h the reaction mixture was worked up in the normal fashion to providethe desired crude amine.

To a stirred solution of the crude amine in THF (3 mL) at roomtemperature was added sat. sodium bicarbonate (3 mL) followed by a dropwise addition of 20% phosgene in toluene (0.115 mL). After stirring for16 h at room temperature, the reaction was worked up in the normalfashion and purified using silica gel chromatography to provide 0.0.81 gof the desired urea.

To a stirred solution of the urea (0.081 g) in degassed chloroform (2mL) at 0° C. was added phenylsilane (0.044 mL) followed bytetrakis(triphenylphosphine) palladium (0) (0.115 mL). After stirringfor 1 h, the reaction was worked up in the normal fashion and purifiedusing DEAE cellulose ion exchange chromatography, then silica gelchromatography followed by SP Sephadex cation exchange to provide 0.054g of the ER-805718.

Reaction Scheme for Phosphate Triester Analogues (Formula II)

Persons familiar with the art can easily envision the preparation of thephosphate triester analogues depicted in Formula II by altering thephosphorylating reagent 11 that will accommodate the new structureslisted. As exemplified in the Scheme below, replacing theallyl-protecting group with the appropriately functionalized substituentprovides the desired structure by normal synthetic processes. In generalthe altered phosphorylating reagent is prepared stepwise by the additionof N-Boc-1-amino-2-ethanol to phosphorus trichloride in the presence ofpyridine. After transforming the dichlorophosphorylmonoester to theactivated bis(diisoproplyamide), the appropriately functionalizedalcohol in the presence of tetrazole is added to provide the desiredprotected functionality or a precursor thereof. The alteredphosphorylating reagent is then used in place of the originalphosphorylating reagent 11 described in the general experimental.Instead of deprotection of the allyl group used for the typicalsynthetic route, the new functionality incorporated into the structureis deprotected by methods available to persons familiar to the art toprovide the alternative desired product listed.

Reaction Scheme for Quaternary Amine Analogues (Formula III)

Persons familiar with the art can easily envision the preparation ofquaternary amine compounds. As exemplified in the Scheme below,oxidation of an alcohol to an aldehyde, reductive animation with theappropriately functionalized amine, followed by protection of theensuing secondary amine with a protecting group such as Fmoc providesthe desired protected intermediate. Selective deprotection of theBoc-group on the primary amine followed by condensation with theappropriate linker such as phosgene provides the protected dimer. Thefinal desired product can be produced by the deprotection of thesecondary amine followed by dialkylation of the amine in the presence ofan simple alkyl halide, such as methyl iodide. The product is purifiedby cation exchange chromatography using CM-Sephadex using dilute HCl asthe eluting counter ion, followed by silica gel chromatography, and thenanion exchange with SP-Sephadex containing the appropriate anioniccounter ion using similar elution solvents as described in the previousexperimentals.

BIOLOGICAL EXAMPLES Example 7 Induction of Cytokines In Vitro

A. Assays in Human Whole Blood

The most readily available human system to test compound activity onmonocytes/macrophages is in whole blood. Various concentrations ofcompounds of the invention were added as 10× stocks in 50 μl of Ca⁺⁺,Mg⁺⁺-free Hank's balanced salt solution (HBSS) followed by 50 μl of HBSSinto 400 μl of heparinized whole blood obtained from normal volunteers(18-51 years old; 110-230 lb.) into the wells of plastic assay plates,for a total volume of 500 μl/well (final concentration of whole bloodwas 80%). After a 3-hour incubation with gentle shaking at 37° C. in a5% CO₂ atmosphere, the assay plates were centrifuged at 1000×g for 10min. at 4° C. and plasma was drawn off and frozen at −80° C. Plasmasamples were analyzed for TNF-alpha, IL-10, and IL-12 by ELISA (GenzymeCorp., Cambridge, Mass.). Each assay point was tested in triplicate.

As shown in FIG. 1, compounds such as 100, 184 and 186 stimulateblood-borne cells to release TNF-alpha. This stimulatory activity can becompared to that of 10 ng/ml endotoxin (or LPS) present in similarincubations in the same assay. As shown in Table 1, activity ofcompounds (tested at 10 μM) ranges from inactive (such as compound 110)to compounds demonstrating greater activity than the LPS standard.

B. Cultured Human Cell Lines

Similar results can be obtained when compounds of the invention aretested in a cell-culture model. In this assay, compounds of theinvention are tested for their ability to stimulate secretion ofalkaline phosphatase from THP-1 cells that have been transfected withthe gene for secreted alkaline phosphatase under the control of theTNF-alpha promoter, as described in detail in Goto et al., MolecularPharmacology 49; 860-873 (1996). In this assay, however, the effects ofremoving serum¹—a condition that may more-likely mimic a subcutaneousenvironment—can be evaluated. As shown in FIG. 2 and described in Table1, results from these assays indicate that compounds of the inventionstimulate induction of genes under the control of the TNF-alpha promoterwhen added to cells in the absence as well as the presence of serum.

¹ This is important to determine if serum components such aslipopolysaccharide binding protein are necessary for drug activity.

TABLE 1 Stimulation of cytokine release by compounds in vitro THP-1 cellStimulation (% of Whole Blood compound 100 at (% of LPS at 10 μM)⁽¹⁾ ER# Compound 10 μM) +serum −serum MPL Standard

29⁽²⁾ 112022

131 ± 10.2 (n = 6) 111230

49 111231

17 111232

158 155 225 111233

141 112043

0 112044

0 112047

0 112048

0 24 112049

0 112063

0 112064

50 112065

86 112066

162 330 112071

0 112072

0 112091

0 112092

0 112093

0 112098

0 112049

0 112100

0 112859

0 112860

0 112861

0 113634

0 113635

0 113643

0 113644

0 113651

133 ± 4.4 (n = 4) 215 254 113665

113666

118023

63 019772

69 118989

159 118999

105 119000

60 119001

113 118949

138 119327

165 ± 33 (n = 3) 119328

181 ± 42 (n = 3) 119329

2 ± 2 (n = 2) 119521

103 119522

129 119523

176 803022

164 803045

65 803056

151 ± 42 803058

149 ± 37 (n = 2) 803059

2 803592

15 ⁽¹⁾Response in each assay was compared to 10 μM compound 100 internalstandard which typically induced 2-3 fold increase in TNF-alpha PLAPexpression over basal. ⁽²⁾Tested at @ 5.8 μM.

C. Murine Splenocytes

The ability of compounds to stimulate cytokine release from splenocytescan be assessed in a mouse model. Spleen cells harvested from C57BL/6mice are cultured for 24 hours in RPMI 1640 cell culture mediumcontaining 5% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mlpenicillin/streptomycin and 50 μM beta-mercaptoethanol, variousconcentrations of test compound for 20-24 hours, after which the cellculture supernatant is tested for the presence of cytokines.

Spleen cells harvested from mice were cultured for 24 house with testcompound and the supernatant was tested for release of cytokines. Asshown in FIGS. 3 and 4, the release of cytokines such as IL-10 andinterferon-gamma from splenocytes is stimulated by compounds such as104, 106, 124, 160, and 162.

These assays utilized a heterogeneous population of cells derived fromthe spleen. This makes it possible that cytokine induction can be causedboth by direct effects of test compounds on cells and through moreindirect stimulation of cytokine “cascades” where the release of acytokine by one type of cell can induce release of other cytokines inother cells present in the same media. It is possible that this cytokine“milieu” is responsible for part of this robust immune responses.

Example 8 In Vivo Induction of Antibody Response

The most critical test of adjuvant activity is the determination if theaddition of a compound to an antigen preparation increases immuneresponse by elevating the level of antibodies generated to that antigenwhen administered to a living animal.

Initial experiments involved the injection of mice (Balb/c) withcompounds of the invention plus a peptide conjugated to a carrier suchas keyhole limpet hemocyanin. The peptide chosen for these studies is apeptide (P18) that corresponds to amino acids 308-322 of the V3 loop ofHIV IIIB gp120 protein. The P18 21aa peptide, corresponding to aminoacids 308-322 of the V3 loop of HIV IIIB gp120 protein, has beenreported to be immunogenic. This peptide with glycine/alanine/glycinespacer residues plus an amino terminal cysteine residue was synthesizedby Genosys (Woodlands Tex.). The peptide sequence is as follows:CGAGIRIQRGPGRAFVTIGKG with the underlined amino acids representing thenative sequence. The peptide was isolated to >80% purity using HPLC bythe supplier. This peptide was coupled via the cysteine residue tobovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) usingmaleimide activated conjugation (Pierce Immunochemical; cat#77107). TheKLH conjugated peptide was used as the immunogen and the BSA conjugateas the screening target antigen for PI 1 specific antibodies. Theindicated amount of KLH-P18 conjugate, was routinely used along with 300μg of test compound, Alum or PBS was injected at 2 or 3 week intervals(as indicated), into male Balb/c mice (Charles River Laboratories)approximately 6-8 weeks old (18-25 g). All injections were subcutaneousat the back of the neck with 200 μl of a mixture of antigen plusadjuvant in PBS administered every two weeks (three weeks forpolysaccharides or influenza) for a total of three injections. Mice werebled one-or two weeks post 2^(nd) and 3^(rd) injections. Sample bleedsare designated as to when taken (i.e. secondary bleed is one week afterthe second protein injection or two weeks after the secondpolysaccharide injections, tertiary bleed is after the third injectionof antigen/adjuvant. Blood was collected after nicking the tail vein anddrops collected into Becton Dickinson brand microtainer serun separatortubes. Serum was separated from the red cells by microcentrifugation andtested by ELISA for antigen specific IgG levels.

Immune response to the peptide can be tested by enzyme-linkedimmunosorbent assay (ELISA), which can quantitate levels of serumantibody that bind to P18 peptide conjugated to anothernon-cross-reacting protein such as bovine serum albumin (P18-BSA) andcoated onto an ELISA plate.

As shown in FIG. 5 and Tables 2 and 3, mice injected with the variouscompounds along with KLH-P18 antigen demonstrated greater response(higher levels of antibody) than those injected with the P18-KLH peptideconjugate alone.

TABLE 2 Stimulation of antibody generation to P18 peptide by compoundsAverage Serum anti-P18 IgG Concentration¹ ER # Compound (fold increaseover no adjuvant) 112022

6.7 112065

19.4 112066

39.2 ¹Concentration of IgG assayed at the tertiary bleed. Average IgGfor serum from 5 mice that were injected with 300 μg compound and 5 μgKLH-P18 conjugate antigen as described in the Methods section.Antigen-specific ELISAs were performed as described in the MethodsSection with Costar EIA/RIA plates coated with 50 μl of 5 μg/ml BSA-P18conjugate in PBS.

TABLE 3 Stimulation of antibody generation to P18 peptide by compoundsAverage Serum anti-P18 IgG Concentration¹ Compound # Compound (foldincrease over no adjuvant) 111232

7.6 112066

13.4* 113651

10.5* 118989

4.35* 119327

16.5* 119328

26.8* ¹Concentration of IgG assayed at the tertiary bleed. Average IgGfor serum from 5 mice that were injected with 300 μg compound andantigen (below) as described in the Methods section. Antigen-specificELISAs were performed as described in the Methods Section with CostarEIA/RIA plates coated with 50 μl of 5 μg/ml BSA-P18 conjugate in PBS. Ascomparison, addition of # Alum increased IgG levels 7.4- fold overPBS/antigen alone. Antigen used: primary: 1 μg KLH-conjugated P18peptide. 2° and 3° boosts: 0.5 μg KLH-conjugated P18 peptide *p < 0.05by Student's two-tailed t-test (unequal variance) compared to the PBS +antigen group.

Adjuvant activity has also been obtained with compounds of the inventionwhen tested with other antigens. Compounds such as 100, 116, 126, 160,184, and 186 can stimulate antigen-specific antibody production by up to26.8-fold (Table 4) to influenza X-31 antigen. Increases in response arealso seen when tetanus toxoid (FIG. 6) and menningococcal Cpolysaccharide (Table 5) are used as challenge antigens. In the assays,1 μg of meningococcal C PS or 1.5 μg tetanus toxoid or 5 μg of influenzaX31 (SPAFAS laboratories) were used. Tetanus toxoid from AccurateChemical (cat #sstettox) was used as a challenge antigen while thepurified toxoid from List Biologicals (cat #191) was used as targetantigen for the ELISA assay.

TABLE 4 Stimulation of antibody generation to Influenza X31 by compoundsAverage Serum anti-Influenza X-31 IgG Concentration¹ ER # Compound (foldincrease over no adjuvant) 112022

1.7 112048

5.4 112066

2.3 113651

1 119327

7.85 119328

26.8 ¹Concentration of IgG assayed at the tertiary bleed. Average IgGfor serum from 5 mice that were injected with 300 μg compound and 5 μgantigen as described in the Methods section. Antigen-specific ELISAswere performed as described in the Methods Section with Costar EIA/RIAplates coated with 50 μl of 10 μg/ml Influenza X-31 antigen in 0.5 Msodium carbonate buffer pH 9.6. As comparison, addition of Alumincreased IgG levels 3.5- fold over PBS/antigen alone.

TABLE 5 Stimulation of antibody generation to Menningococcalpolysaccharide by compounds Average Serum anti-Menningococcal PS IgGConcentration¹ ER # Compound (fold increase over no adjuvant) 112022

8.3 112048

1.8 112066

12.9 113651

18.3 119327

15.8 ¹Concentration of IgG assayed at the secondary bleed. Average IgGfor serum from 5 mice that were injected with 300 μg compound and 1 μgantigen as described in the Methods section. Antigen-specific ELISAswere performed as described in the Methods Section with Costar EIA/RIAplates coated with 50 μl of 5 μg/ml meningococcal PS in PBS plusmethylated human serum albumin as described Gheesling et al. J. ClinMicrobiol.32; 1475-82(1994). In comparison, addition of Alum increasedIgG levels 2.3- fold over PBS/antigen alone.

Influenza virus X-31 was purchased from SPAFAS (Storrs, Conn.) and wasinactivated and confirmed to be inactive [Payne et al. Vaccine 16; 92-98(1998)] by the supplier. Menningococcal C polysaccharide (PS) wassupplied by Pasteur Merrieur Connaught (Swiftwater Pa.). Methylatedhuman albumin can be obtained according to the methods described byGheesling et al. J. Clin Microbiol.32; 1475-82 (1994).

For the preparation of antigen/adjuvant mixtures, lyophilized testcompounds were reconstituted to 2 mg/ml with phosphate buffered saline(PBS; cat # P-3813; Sigma Chemical Co, St Louis Mo.) and sonicated in achilled water bath for two minutes. Monophosphoryl Lipid A, MPL, (RibiImmunochemical) was reconstituted to 2 mg/ml with sterile water forinjection, incubated at 50° C. for 15 minutes and then sonicated asabove. Imject^(R) Alum, purchased from Pierce Immunochemical, was usedaccording to manufacturer's guidelines, and comprised approximately20-30% of the injection volume. Indicated amounts of antigen, diluted inPBS, were mixed with the compounds, MPL, or Alum such that the finalconcentration of the compound or MPL was 300 μg (unless otherwise noted)in the 200 μl injection volume. The mixtures were incubated at roomtemperature for 40 minutes with continuous shaking prior to injection.

Antigen specific IgG levels were monitored by direct ELISA where antigenwas passively coated onto 96 well Costar EIA/RIA plates. Plates werecoated with 50 μl/well of the indicated antigen and incubated overnight(ON) at 4° C. and washed 3× with PBS+0.05% tween 20 (PBS-t) in anautomated plate washer. Plates were then blocked with 200 μl/well of0.5% gelatin in PBS for 1 hr at room temperature (RT) and washed 3× withPBS-t. Mouse sera was diluted in PBS-t plus 0.3% BSA and 100 μl ofvarious dilutions were added, in duplicate to the antigen coated wells(or BSA coated wells as a control) and incubated at RT for 1 hr. andagain washed 3× with PBS-t. Biotinylated goat anti-mouse IgG (SouthernBiotechnology Associates Inc., Birmingham Ala., cat #1031-08) wasdiluted 1:5000 in PBS-t and 100 μl/well was applied and incubated at RTfor 1 hr, washed 3× with PBS-T and followed by the addition of 1:10,000streptavidin-horseradish peroxidase conjugate (Southern BiotechnologyAssociates Inc.) in PBS-t for 30 minutes at RT and again washed 3× withPBS-t. Wells were then incubated in 100 μL TMB substrate (Kirkegaard andPerry Labs) for 5 minutes. Color development was stopped with theaddition of an equal volume of 1M phosphoric acid and the absorbance wasread at 450 nm on a Titertek Multiscan plate reader with Deltasoftsoftware analysis package.

For relative quantitation of antigen-specific IgG levels, curves werecompared to one another by determining the dilution necessary to obtaina fixed amount of antibody-generated color. In some cases, a total IgGassay using an anti-FAb-specific reagent to capture known amounts ofpurified IgG (purchased from Southern Biotech.) as an IgG standard curvewas run in conjunction with the direct ELISA on the BSA-P18 conjugate.The anti-FAb reagent orients the purified IgG in a manner similar to howan antibody would bind to an antigen through the FAb region. This allowsdetection of bound antibody by the same reagents used to measureantigen-specific capture of antibodies. The same reagent solutions usedfor detection of the antibodies bound to BSA-P18 conjugate, namelybiotinylated anti-IgG (Fc-specific) followed with HRP-streptavidin, weresimultaneously applied to the anti-FAb total IgG quantitative assay andto the antigen-specific assay. Hence, the signal from the binding of thepurified IgG standard curve is equivalent to that generated to equalamounts of IgG bound in the anti-target antigen assay. The amount ofantibody in the serum is then interpolated from the purified IgGstandard using a 4-parameter curve fit (DeltaSoft 3 software package).

TABLE 6 WB ED₅₀ vs. % of LPS at 10 ng/ml WB ED₅₀ vs LPS @ ER # Structure10 ng/ml MPL Standard

>>10 μm 112022

0.696 μm 111230

111231

0.29 μm 111232

111233

112043

112044

112047

112048

>>10 μM 112049

112063

112064

112065

0.25 μM 112066

0.04 μM 112071

112072

112091

112092

112093

112098

112099

112100

112859

112860

112861

113634

113635

113643

113644

113651

0.70 μM 113665

113666

118023

019772

118989

0.1 μM 118999

119000

119001

1.23 μM 118949

119327

0.015 μM 119328

>>10 μM 119329

119521

119522

119523

803022

0.06 μM 803028

803045

803056

803058

0.022 μM 803059

0.89 μM 803592

803596

803597

803598

803599

803613

803731

>10 μM 803732

0.85 μM 803733

0.70 μM 803751

803783

803784

803789

0.10 μM 804053

1.34 μM 804057

0.008 μM 804058

0.03 μM 804059

>10 μM 804061

2.5 μM 804097

0.3 μM 804121

0.46 μM 804130

0.66 μM 804221

2.2 μM 804222

0.008 μM 804252

400 nM (576-021) + EtOH 804253

>10 μM 804281

0.45 μM 804313

0.014 μM 804339

1.06 μM 804372

0.4 μM 804442

0.007 μM 804503

0.35 μM 804558

0.16 μM 804596

>10 μM 804674

1.2 μM 804678

0.018 μM 804679

0.53 μM 804680

0.015 μM 804732

<0.001 μM 804764

0.015 μM 804772

0.008 μM 804947

>>10 μM

Table 7 below contains the compound number as referenced herein to thecorresponding ER number.

TABLE 7 Correspondence of Compound Nos. to ER Nos. Compound # ER #Compound # ER #  16 112048 152 113634  31 803058 154 113635  48 803733156 113643  50 803022 158 113644  62 803789 160 113651  72 803592 164113665 100 112022 166 113666 102 111230 168 118023 104 111231 170 019772106 111232 172 118989 108 111233 176 118999 110 112043 178 119000 112112047 180 119001 114 112047 182 118949 116 112048 184 119327 118 112049186 119328 120 112063 188 119329 122 112064 190 119521 124 112065 192119522 126 112066 194 119523 128 112071 196 803022 130 112072 198 803045132 112091 200 803056 134 112092 202 803058 136 112093 204 803059 138112098 206 803592 140 112099 142 112100 146 112859 148 112860 150 112861

What is claimed is:
 1. A compound of formula I, II or III:

wherein for each of formula I, II or III: R¹ is selected from the groupconsisting of (a) C(O); (b) C(O)—C₁₋₄ alkyl-C(O), wherein said C₁₋₁₄alkyl is optionally substituted with hydroxy, C₁₋₅ alkoxy, C₁₋₅alkylenedioxy, C₁₋₅ alkylamino, or C₁₋₅ alkyl-aryl, wherein said arylmoiety of said C₁₋₁₅-alkyl-aryl is optionally substituted with C₁₋₅alkoxy, C₁₋₅ alkyl amino, C₁₋₅ alkoxy-amino, C₁₋₅ alkylamino-C₁₋₅alkoxy, —O—C₁₋₅ alkylamino-C₁₋₅ alkoxy, —O—C₁₋₅ alkylamino-C(O)—C₁₋₅alkyl C(O)OH, —O—C₁₋₅ alkylamino-C(O)-C₁₋₅ alkyl-C(O)—C₁₋₅ alkyl; (c) C₂to C₁₅ straight or branched chain alkyl optionally substituted withhydroxy or alkoxy; and (d) —C(O)—C₆₋₁₂ arylene-C(O)— wherein saidarylene is optionally substituted with hydroxy, halogen, nitro or amino;a and b are independently 0, 1, 2, 3 or 4; d, d′, d″, e, e′ and e″ areindependently an integer from 0 to 4; X¹, X², Y¹ and Y² areindependently selected from the group consisting of null, oxygen, NH andN(C(O)C₁₋₄ alkyl), and —N(C₁₋₄ alkyl)-; W¹ and W² are independentlyselected from the group consisting of carbonyl, methylene, sulfone andsulfoxide; R² and R⁵ are independently selected from the groupconsisting of: (a) C₂ to C₂₀ straight chain or branched chain alkylwhich is optionally substituted with oxo, hydroxy or alkoxy, (b) C₂ toC₂₀ straight chain or branched chain alkenyl or dialkenyl which isoptionally substituted with oxo, hydroxy or alkoxy; (c) C₂ to C₂₀straight chain or branched chain alkoxy which is optionally substitutedwith oxo, hydroxy or alkoxy; (d) —NH—C₂ to C₂₀ straight chain orbranched chain alkyl, wherein said alkyl group is optionally substitutedwith oxo, hydroxy or alkoxy; and (e)

wherein Z is selected from the group consisting of O and NH, and M and Nare independently selected from the group consisting of C₂ to C₂₀straight chain or branched chain alkyl, alkenyl, alkoxy, acyloxy,alkylamino, and acylamino; R¹ and R⁶ are independently selected from thegroup consisting of C₂ to C₂₀ straight chain or branched chain alkyl oralkenyl optionally substituted with oxo or fluoro; R⁴and R⁷ areindependently selected from the group consisting of C(O)C₂ to C₂₀straight chain or branched chain alkyl or alkenyl; C₂ to C₂₀ straightchain or branched chain alkyl; C₂ to C₂₀ straight chain or branchedchain alkoxy; C₂ to C₂₀ straight chain or branched chain alkenyl;wherein said alkyl, alkenyl or alkoxy groups can be independently andoptionally substituted with hydroxy, fluoro or C₁ to C₅ alkoxy; G¹, G²,G³ and G⁴ are independently selected from the group consisting ofoxygen, methylene, amino, thiol, —O—C(O)—, —NHC(O)—, —C(O)NH—, and—N(C(O)C₁₋₄ alkyl)-; or G²R⁴ or G⁴R⁷ may together be a hydrogen atom orhydroxyl; or a pharmaceutically acceptable salt thereof; and wherein forFormula II: a′ and b′ are independently 2, 3, 4, 5, 6, 7, or 8,preferably 2; Z¹ is selected from the group consisting of —OP(O)(OH)₂,—P(O)(OH)₂, —OP(O)(OR⁸)(OH) where R⁸ is a C₁-C₄ alkyl chain, —OS(O)₂OH,—S(O)₂OH—, —CO₂H, —OB(OH)₂, —OH, —CH₃, —NH₂, NR⁹ ₃ where R⁹ is a C₁-C₄alkyl chain; Z² is —OP(O)(OH)₂, —P(O)(OH)_(2′), —OP(O)(OR¹⁰)(OH) whereR¹⁰ is a C₁-C₄ alkyl chain, —OS(O)₂OH, —S(O)₂OH, CO₂H, —OB(OH)₂, —OH,CH₃, —NH₂, —NR¹¹, where R¹¹ is C₁-C₄ alkyl chain; and wherein forFormula III: R¹² is selected from H and a C₁-C₄ alkyl chain; or apharmaceutical salt thereof, with the proviso that the compounds offormula I, II or III are not


2. The compound of claim 1, wherein R¹ is selected from the groupconsisting of C(O) and C(O)—C₁₋₁₄alkyl-C(O).
 3. The compound of claim 1,wherein each of a and b are
 2. 4. The compound of claim 1, wherein a′and b′ are
 2. 5. The compound of claim 1, wherein X¹ and Y¹ are both NH.6. The compound of claim 1, wherein d and e are
 1. 7. The compound ofclaim 1, wherein G²R⁴ and G⁴R⁷ are both hydroxy.
 8. An immunologicaladjuvant formulation comprising the compound of claim
 13. 9. A vaccineformulation comprising an antigen and the compound of claim
 1. 10. Theformulation of claim 9, wherein the compound of and the antigen arecovalently bonded through an amino, carbonyl, hydroxyl, or phosphatemoiety of the compound.
 11. The formulation of claim 9, wherein R¹ is aC(O) or C(O)—C₃₋₁₄alkyl-C(O), and wherein the compound and the antigenare covalently bonded to a carbonyl moiety of said C¹ carbonyl of saidC(O) or C(O)—C₃₋₁₄alkyl-C(O).
 12. A method for stimulating an immuneresponse in an animal in need thereof, comprising administering aneffective amount of an antigen and an effective amount for stimulatingan immune response to said antigen of the compound of claim
 1. 13. Acompound of formula I:

wherein R¹ is selected from the group consisting of (a) C(O); (b)C(O)—C₁₋₄ alkyl-C(O), wherein said C₁₋₁₄ alkyl is optionally substitutedwith hydroxy, C₁₋₅ alkoxy, C₁₋₅ alkylenedioxy, C₁₋₅ alkylamino, or C₁₋₅alkyl-aryl, wherein said aryl moiety of said C₁₋₁₅-alkyl-aryl isoptionally substituted with C₁₋₅ alkoxy, C₁₋₅ alkyl amino, C₁₋₅alkoxy-amino, C₁₋₅ alkylamino-C₁₋₅ alkoxy, —O—C₁₋₅ alkylamino-C₁₋₅alkoxy, —O—C₁₋₅ alkylamino-C(O)—C₁₋₅ alkyl C(O)OH, —O—C₁₋₅alkylamino-C(O)—C₁₋₅ alkyl-C(O)—C₁₋₅ alkyl; (c) C₂ to C₁₅ straight orbranched chain alkyl optionally substituted with hydroxy or alkoxy; and(d) —C(O)—C₆₋₁₂ arylene-C(O)— wherein said arylene is optionallysubstituted with hydroxy, halogen, nitro or amino; a and b areindependently 0, 1, 2, 3 or 4; d, d′, d″, e, e′ and e″ are independentlyan integer from 0 to 4; X¹, X², Y¹ and Y² are independently selectedfrom the group consisting of null, oxygen, NH and —N(C(O)C₁₋₄ alkyl)-,and —N(C₁₋₄ alkyl)-; W¹ and W² are independently selected from the groupconsisting of carbonyl, methylene, sulfone and sulfoxide; R² and R⁵ areindependently selected from the group consisting of: (a) C₂ to C₂₀straight chain or branched chain alkyl which is optionally substitutedwith oxo, hydroxy or alkoxy, (b) C₂ to C₂₀ straight chain or branchedchain alkenyl or dialkenyl which is optionally substituted with oxo,hydroxy or alkoxy; (c) C₂ to C₂₀ straight chain or branched chain alkoxywhich is optionally substituted with oxo, hydroxy or alkoxy; (d) —NH—C₂to C₂₀ straight chain or branched chain alkyl, wherein said alkyl groupis optionally substituted with oxo, hydroxy or alkoxy; and (e)

wherein Z is selected from the group consisting of O and NH, and M and Nare independently selected from the group consisting of C₂ to C₂₀straight chain or branched chain alkyl, alkenyl, alkoxy, acyloxy,alkylamino, and acylamino; R¹ and R⁶ are independently selected from thegroup consisting of C₂ to C₂₀ straight chain or branched chain alkyl oralkenyl optionally substituted with oxo or fluoro; R⁴ and R⁷ areindependently selected from the group consisting of C(O)C₂ to C₂₀straight chain or branched chain alkyl or alkenyl; C₂ to C₂₀ straightchain or branched chain alkyl; C₂ C₂₀ straight chain or branched chainalkoxy; C₂ to C₂₀ straight chain or branched chain alkenyl; wherein saidalkyl, alkenyl or alkoxy groups can be independently and optionallysubstituted with hydroxy, fluoro or C₁ to C₅ alkoxy; G¹, G², G³ and G⁴are independently selected from the group consisting of oxygen,methylene, amino, thiol, —OC(O)—, —NHC(O)—, —C(O)NH—, and —N(C(O)C₁₋₄alkyl)-; or G²R⁴ or G⁴R⁷ may together be a hydrogen atom or hydroxyl; ora pharmaceutically acceptable salt thereof; or a pharmaceutical saltthereof, with the proviso that the compounds of formula I are not


14. The compound of claim 13, wherein R¹ is selected from the groupconsisting of C(O) and C(O)—C₁₋₄alkyl-C(O).
 15. The compound of claim13, wherein each of a and b are
 2. 16. The compound of claim 13, whereinX¹ and Y¹ are both NH.
 17. The compound of claim 13, wherein d and eare
 1. 18. An immunological adjuvant formulation comprising the compoundof claim
 13. 19. A vaccine formulation comprising an antigen and thecompound of claim
 13. 20. The formulation of claim 19, wherein thecompound of formula I and the antigen are covalently bonded through anamino, carbonyl, hydroxyl, or phosphate moiety of the compound offormula I.
 21. The formulation of claim 19, wherein R¹ is a C(O) orC(O)—C₃₋₁₄alkyl-C(O), and wherein compound of formula I and antigen arecovalently bonded to a carbonyl moiety of said C₁ carbonyl of said C(O)or C(O)—C₃₋₁₄alkyl-C(O).
 22. A method for stimulating an immune responsein an animal in need thereof, comprising administering an effectiveamount of an antigen and an effective amount for stimulating an immuneresponse to said antigen of the compound of claim
 13. 23. A compound offormula VI:

wherein R¹ is selected from the group consisting of C₁ carbonyl, C₂ toC₁₅ dicarbonyl optionally substituted with hydroxy or alkoxy, and C₂ toC₁₅ straight or branched chain alkyl optionally substituted with hydroxyor alkoxy; wherein each of a and b is independently an integer from 2 to4; wherein each of d and e is independently an integer from 1 to 4;wherein each of X and Y is independently selected from the groupconsisting of oxygen, and NH; wherein R² and R⁵ are independentlyselected from the group consisting of: (a) C₂ to C₂₀ straight chain orbranched chain alkyl, wherein said C₂ C₂₀ straight chain or branchedchain alkyl is optionally substituted with hydroxy or alkoxy, (b) C₂ toC₂₀ straight chain or branched chain alkenyl, wherein said C₂ C₂₀straight chain or branched chain alkenyl is optionally substituted withhydroxy or alkoxy; (c) CH₂-alkyl carbonyl; (d) CH₂-alkenyl carbonyl; and(e)

wherein Z is selected from the group consisting of O and NH, and M and Nare independently selected from the group consisting of C₂ to C₂₀straight chain or branched chain alkyl, alkenyl, alkoxy, acyloxy,alkylamino, and acylamino; wherein R³ and R⁶ are independently selectedfrom the group consisting of C₂ to C₂₀ straight chain or branched chainalkyl and C₂ to C₂₀ straight chain or branched chain alkenyl; wherein R⁴and R⁷ are independently selected from the group consisting of hydrogen,C₂ to C₂₀ straight chain or branched chain alkyl, C₂ to C₂₀ straightchain or branched chain alkenyl, C₂ to C₂₀ straight chain or branchedchain alkyl carbonyl, and C₂ to C₂₀ straight chain or branched chainalkenyl carbonyl wherein said C₂ to C₂₀ straight chain or branched chainalkyl and C₂ to C₂₀ straight chain or branched chain alkenyl can beindependently and optionally substituted with hydroxy or alkoxy; or apharmaceutically acceptable salt thereof; with the proviso that thecompound of formula IV is not


24. The compound of claim 23, wherein R¹ is C(O).
 25. The compound ofclaim 23, wherein d and e are
 1. 26. An immunological adjuvantformulation comprising the compound of claim
 23. 27. A vaccineformulation comprising an antigen and the compound of claim
 23. 28. Theformulation of claim 27, wherein the compound of formula IV and theantigen are covalently bonded through an amino, carbonyl, hydroxyl, orphosphate moiety of the compound of formula IV.
 29. The formulation ofclaim 27, wherein R¹ is C(O) and wherein the compound of formula IV andantigen are covalently bonded to said C(O).
 30. A method for stimulatingan immune response in a patient in need thereof, comprisingadministering an effective amount of an antigen and an effective amountfor stimulating an immune response to said antigen of the compound ofclaim
 23. 31. A compound of formula V:


32. An immunological adjuvant formulation comprising the compound ofclaim
 31. 33. A vaccine formulation comprising an antigen and thecompound of claim
 31. 34. The formulation of claim 33, wherein thecompound of formula V and the antigen are covalently bonded through anamino, carbonyl, or phosphate moiety of the compound of formula V. 35.The formulation of claim 33, wherein the compound of formula V and theantigen are covalently bonded to the carbonyl moiety located between thephosphates.
 36. A method for stimulating an immune response in a patientin need thereof, comprising administering an effective amount of anantigen and an effective amount for stimulating an immune response tosaid antigen of the compound of claim 31.